Methods and compositions for improved F-18 labeling of proteins, peptides and other molecules

ABSTRACT

The present application discloses compositions and methods of synthesis and use of  68 Ga,  18 F or  19 F labeled molecules of use in PET or MRI imaging. Preferably, the  18 F or  19 F is conjugated to a targeting molecule by formation of a complex with a group IIIA metal and binding of the complex to a chelating moiety, which may be directly or indirectly attached to the targeting molecule. In other embodiments, the  68 Ga,  18 F or  19 F labeled moiety may comprise a targetable construct used in combination with a bispecific antibody to target a disease-associated antigen. In more preferred embodiments, a chelating moiety or targetable construct may be conjugated to a targeting molecule, such as an antibody or antibody fragment.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/958,889, filed Dec. 2, 2010, which was a continuation-in-part of U.S.patent application Ser. No. 12/433,212 (now issued U.S. Pat. No.8,153,100), filed Apr. 30, 2009, which was a continuation-in-part ofU.S. patent application Ser. No. 12/343,655 (now issued U.S. Pat. No.7,993,626), filed Dec. 24, 2008, which was a continuation-in-part ofU.S. patent application Ser. No. 12/112,289 (now issued U.S. Pat. No.7,563,433), filed Apr. 30, 2008, which was a continuation-in-part ofU.S. patent application Ser. No. 11/960,262 (now issued U.S. Pat. No.7,597,876), filed Dec. 19, 2007, which claimed the benefit under 35U.S.C. 119(e) of provisional U.S. Patent Application 60/884,521, filedJan. 11, 2007. This application claims the benefit under 35 U.S.C.119(e) of provisional U.S. Patent Application Nos. 61/266,773, filedDec. 4, 2009; 61/302,280, filed Feb. 8, 2010; 61/316,125, filed Mar. 22,2010; 61/347,486, filed May 24, 2010; 61/381,720, filed Sep. 10, 2010and 61/388,268, filed Sep. 30, 2010. Each priority application isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 2, 2010, isnamed IMM31US7.txt and is 16,944 bytes in size.

FIELD

The present invention concerns methods of labeling peptides or othermolecules with ¹⁸F or ¹⁹F that are of use, for example, in PET or NMR invivo imaging. Preferably, the ¹⁸F or ¹⁹F is attached as a conjugate[complex] with aluminum or another metal via a chelating moiety, whichmay be covalently linked to a protein, peptide or other molecule. Usingthe techniques described herein, ¹⁸F— or ¹⁹F-labeled molecules of highspecific activity may be prepared in 30 minutes or less and are suitablefor use in imaging techniques without the need for HPLC purification ofthe labeled molecule. Labeling may occur in a saline medium suitable fordirect use in vivo. In alternative embodiments an organic solvent may beadded to improve the efficiency of labeling. The labeled molecules arestable under in vivo conditions, although for certain purposes, such askit formulations, a stabilizing agent such as ascorbic acid, trehalose,sorbitol or mannitol may be added. In other alternative embodiments, achelating moiety may be preloaded with aluminum and lyophilized forstorage, prior to labeling with ¹⁸F or ¹⁹F.

BACKGROUND

Positron Emission Tomography (PET) has become one of the most prominentfunctional imaging modalities in diagnostic medicine, with very highsensitivity (fmol), high resolution (4-10 mm) and tissue accretion thatcan be adequately quantitated (Volkow et al., 1988, Am. J. Physiol.Imaging 3:142). Although [¹⁸F]2-deoxy-2-fluoro-D-glucose ([¹⁸F]FDG) isthe most widely used functional imaging agent in oncology (Fletcher etal., 2008, J. Nucl. Med. 49:480), there is a keen interest in developingother labeled compounds for functional imaging to complement and augmentanatomic imaging methods (Torigian et al., 2007, CA Cancer J. Clin.57:206), especially with the hybrid PET/computed tomography systemscurrently in use. Thus, there is a need to have facile methods ofconjugating positron-emitting radionuclides to various molecules ofbiological and medical interest.

Peptides or other small molecules can be labeled with the positronemitters ¹⁸F, ⁶⁴Cu, ¹¹C, ⁶⁶Ga, ⁶⁸Ga, ⁷⁶Br, ^(94m)Tc, ⁸⁶Y, and ¹²⁴I. Alow ejection energy for a PET isotope is desirable to minimize thedistance that the positron travels from the target site before itgenerates the two 511 keV gamma rays that are imaged by the PET camera.Many isotopes that emit positrons also have other emissions such asgamma rays, alpha particles or beta particles in their decay chain. Itis desirable to have a PET isotope that is a pure positron emitter sothat any dosimetry problems will be minimized. The half-life of theisotope is also important, since the half-life must be long enough toattach the isotope to a targeting molecule, analyze the product, injectit into the patient, and allow the product to localize, clear fromnon-target tissues and then image. If the half-life is too long thespecific activity may not be high enough to obtain enough photons for aclear image and if it is too short the time needed for manufacturing,commercial distribution and biodistribution may not be sufficient. ¹⁸F(β⁺ 635 keV 97%, t_(1/2) 110 min) is one of the most widely used PETemitting isotopes because of its low positron emission energy, lack ofside emissions and suitable half-life.

Conventionally, ¹⁸F is attached to compounds by binding it to a carbonatom (Miller et al., 2008, Angew Chem Int Ed 47:8998-9033), butattachments to silicon (Shirrmacher et al., 2007, Bioconj Chem18:2085-89; Hohne et al., 2008, Bioconj Chem 19:1871-79) and boron (Tinget al., 2008, Fluorine Chem 129:349-58) have also been reported. Bindingto carbon usually involves multistep syntheses, including multiplepurification steps, which is problematic for an isotope with a 110-minhalf-life. Current methods for ¹⁸F labeling of peptides typicallyinvolve the labeling of a reagent at low specific activity, HPLCpurification of the reagent and then conjugation to the peptide ofinterest. The conjugate is often repurified after conjugation to obtainthe desired specific activity of labeled peptide.

An example is the labeling method of Poethko et al. (J. Nucl. Med. 2004;45: 892-902) in which 4-[¹⁸F]fluorobenzaldehyde is first synthesized andpurified (Wilson et al, J. Labeled Compounds and Radiopharm. 1990;XXVIII: 1189-1199) and then conjugated to the peptide. The peptideconjugate is then purified by HPLC to remove excess peptide that wasused to drive the conjugation to completion. Other examples includelabeling with succinyl [¹⁸F]fluorobenzoate(SFB) (e.g., Vaidyanathan etal., 1992, Int. J. Rad. Appl. Instrum. B 19:275), other acyl compounds(Tada et al., 1989, Labeled Compd. Radiopharm.XXVII:1317; Wester et al.,1996, Nucl. Med. Biol. 23:365; Guhlke et al., 1994, Nucl. Med. Biol21:819), or click chemistry adducts (Li et al., 2007, Bioconjugate Chem.18:1987). The total synthesis and formulation time for these methodsranges between 1-3 hours, with most of the time dedicated to the HPLCpurification of the labeled peptides to obtain the specific activityrequired for in vivo targeting. With a 2 hr half-life, all of themanipulations that are needed to attach the ¹⁸F to the peptide are asignificant burden. These methods are also tedious to perform andrequire the use of equipment designed specifically to produce thelabeled product and/or the efforts of specialized professional chemists.They are also not conducive to kit formulations that could routinely beused in a clinical setting.

A need exists for a rapid, simple method of ¹⁸F labeling of targetingmoieties, such as proteins or peptides, which results in targetingconstructs of suitable specific activity and in vivo stability fordetection and/or imaging, while minimizing the requirements forspecialized equipment or highly trained personnel and reducing operatorexposure to high levels of radiation. More preferably a need exists formethods of preparing ¹⁸F-labeled targeting peptides of use inpretargeting technologies. A further need exists for prepackaged kitsthat could provide compositions required for performing such novelmethods.

SUMMARY

In various embodiments, the present invention concerns compositions andmethods relating to ¹⁸F- or ¹⁹F-labeled molecules of use for PET or NMRimaging. As discussed herein, where the present application refers to¹⁸F the skilled artisan will realize that either ¹⁸F, ¹⁹F or anothermetal-binding radionuclide, such as ⁶⁸Ga, may be utilized. In anexemplary approach, the ¹⁸F is bound to a metal and the ¹⁸F-metalcomplex is attached to a ligand on a peptide or other molecule. Asdescribed below, the metals of group IIIA (aluminum, gallium, indium,and thallium) are suitable for ¹⁸F binding, although aluminum ispreferred. Lutetium may also be of use. The metal binding ligand ispreferably a chelating agent, such as NOTA, NETA, DOTA, DTPA and otherchelating groups discussed in more detail below. Alternatively, one canattach the metal to a molecule first and then add the ¹⁸F to bind to themetal.

The skilled artisan will realize that virtually any delivery moleculecan be attached to ¹⁸F for imaging purposes, so long as it containsderivatizable groups that may be modified without affecting theligand-receptor binding interaction between the delivery molecule andthe cellular or tissue target receptor. Although the Examples belowprimarily concern ¹⁸F-labeled peptide moieties, many other types ofdelivery molecules, such as oligonucleotides, hormones, growth factors,cytokines, chemokines, angiogenic factors, anti-angiogenic factors,immunomodulators, proteins, nucleic acids, antibodies, antibodyfragments, drugs, interleukins, interferons, oligosaccharides,polysaccharides, lipids, etc. may be ¹⁸F-labeled and utilized forimaging purposes.

Exemplary targetable construct peptides described in the Examples below,of use for pre-targeting delivery of ¹⁸F or other agents, include butare not limited to IMP 449, IMP 460, IMP 461, IMP 467, IMP 469, IMP 470,IMP 471, IMP 479, IMP 485 and IMP 487, comprising chelating moietiesthat include, but are not limited to, DTPA, NOTA, benzyl-NOTA, alkyl oraryl derivatives of NOTA, NODA-GA, C-NETA, succinyl-C-NETA andbis-t-butyl-NODA.

In certain embodiments, the exemplary ¹⁸F-labeled peptides may be of useas targetable constructs in a pre-targeting method, utilizing bispecificor multispecific antibodies or antibody fragments. In this case, theantibody or fragment will comprise one or more binding sites for atarget associated with a disease or condition, such as atumor-associated or autoimmune disease-associated antigen or an antigenproduced or displayed by a pathogenic organism, such as a virus,bacterium, fungus or other microorganism. A second binding site willspecifically bind to the targetable construct. Methods for pre-targetingusing bispecific or multispecific antibodies are well known in the art(see, e.g., U.S. Pat. No. 6,962,702, the Examples section of which isincorporated herein by reference.) Similarly, antibodies or fragmentsthereof that bind to targetable constructs are also well known in theart, such as the 679 monoclonal antibody that binds to HSG (histaminesuccinyl glycine) or the 734 antibody that binds to In-DTPA (see U.S.Pat. Nos. 7,429,381; 7,563,439; 7,666,415; and 7,534,431, the Examplessection of each incorporated herein by reference). Generally, inpretargeting methods the bispecific or multispecific antibody isadministered first and allowed to bind to cell or tissue targetantigens. After an appropriate amount of time for unbound antibody toclear from circulation, the e.g. ¹⁸F-labeled targetable construct isadministered to the patient and binds to the antibody localized totarget cells or tissues, then an image is taken for example by PETscanning.

In alternative embodiments, molecules that bind directly to receptors,such as somatostatin, octreotide, bombesin, folate or a folate analog,an RGD peptide or other known receptor ligands may be labeled and usedfor imaging. Receptor targeting agents may include, for example, TA138,a non-peptide antagonist for the integrin α_(v)β₃ receptor (Liu et al.,2003, Bioconj. Chem. 14:1052-56). Other methods of receptor targetingimaging using metal chelates are known in the art and may be utilized inthe practice of the claimed methods (see, e.g., Andre et al., 2002, J.Inorg. Biochem. 88:1-6; Pearson et al., 1996, J. Med., Chem.39:1361-71).

The type of diseases or conditions that may be imaged is limited only bythe availability of a suitable delivery molecule for targeting a cell ortissue associated with the disease or condition. Many such deliverymolecules are known. For example, any protein or peptide that binds to adiseased tissue or target, such as cancer, may be labeled with ¹⁸F bythe disclosed methods and used for detection and/or imaging. In certainembodiments, such proteins or peptides may include, but are not limitedto, antibodies or antibody fragments that bind to tumor-associatedantigens (TAAs). Any known TAA-binding antibody or fragment may belabeled with ¹⁸F by the described methods and used for imaging and/ordetection of tumors, for example by PET scanning or other knowntechniques.

Certain alternative embodiments involve the use of click chemistryreactions for attachment of metal-¹⁸F-chelating moieties to molecules.Preferably, the click chemistry involves the reaction of a targetingmolecule such as an antibody or antigen-binding antibody fragment,comprising an activating moiety such as an alkyne, a nitrone or an azidegroup, with a chelating moiety or a carrier molecule attached to one ormore chelating groups and comprising a corresponding reactive moiety,such as an azide, alkyne or nitrone. Where the targeting moleculecomprises an alkyne, the chelating moiety or carrier will comprise anazide, a nitrone or similar reactive moiety. The click chemistryreaction may occur in vitro to form a highly stable, labeled targetingmolecule that is then administered to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are included to illustrate particular embodimentsof the invention and are not meant to be limiting as to the scope of theclaimed subject matter.

FIG. 1. Biodistribution of ¹⁸F-labeled agents in tumor-bearing nude miceby microPET imaging. Coronal slices of 3 nude mice bearing a small,subcutaneous LS174T tumor on each left flank after being injected witheither (A) [¹⁸F]FDG, (B) Al[¹⁸F] IMP 449 pretargeted with theanti-CEA×anti-HSG bsMAb, (C) Al[¹⁸F] IMP 449 alone (not pretargeted withthe bsMAb). Biodistribution data expressed as percent-injected dose pergram (% ID/g) are given for the tissues removed from the animals at theconclusion of the imaging session. Abbreviations: B, bone marrow; H,heart; K, kidney; T, tumor.

FIG. 2. Dynamic imaging study of pretargeted Al[¹⁸F]IMP 449 given to anude mouse bearing a 35-mg LS174T human colorectal cancer xenograft inthe upper flank. The top 3 panels show coronal, sagittal, and transversesections, respectively, taken of a region of the body centering on thetumor's peripheral location at 6 different 5-min intervals over the120-min imaging session. The first image on the left in each sectionalview shows the positioning of the tumor at the intersection of thecrosshairs, which is highlighted by arrows. The animal was partiallytilted to its right side during the imaging session. The bottom 2 panelsshow additional coronal and sagittal sections that focus on a moreanterior plane in the coronal section to highlight distribution in theliver and intestines, while the sagittal view crosses more centrally inthe body. Abbreviations: Cor, coronal; FA, forearms; H, heart; K,kidney; Lv, liver; Sag, sagittal; Tr, transverse; UB, urinary bladder.

FIG. 3. In vivo tissue distribution with ¹⁸F-labeled IMP 468 bombesinanalogue.

FIG. 4. Comparison of biodistribution of ¹⁸F-IMP 466 and ⁶⁸Ga-IMP 466 at2 h p.i. in AR42J tumor-bearing mice (n=5). As a control, mice inseparate groups (n=5) received an excess of unlabeled octreotide todemonstrate receptor specificity.

FIG. 5. Coronal slices of PET/CT scan of ¹⁸F-IMP 466 and ⁶⁸Ga-IMP 466 at2 h p.i. in mice with an s.c. AR42J tumor in the neck. Accumulation intumor and kidneys is clearly visualized.

FIG. 6. Biodistribution of 6.0 nmol ¹²⁵I-TF2 (0.37 MBq) and 0.25 nmol⁶⁸Ga-IMP 288 (5 MBq), 1 h after i.v. injection of ⁶⁸Ga-IMP 288 in BALB/cnude mice with a subcutaneous LS174T and SK-RC52 tumor. Values are givenas means±standard deviation (n=5).

FIG. 7. Biodistribution of 5 MBq FDG and of 5 MBq ⁶⁸Ga-IMP 288 (0.25nmol) 1 hour after i.v. injection following pretargeting with 6.0 nmolTF2. Values are given as means±standard deviation (n=5).

FIG. 8. PET/CT images of a BALB/c nude mouse with a subcutaneous LS174Ttumor (0.1 g) on the right hind leg (light arrow) and a inflammation inthe left thigh muscle (dark arrow), that received 5 MBq ¹⁸F-FDG, and oneday later 6.0 nmol TF2 and 5 MBq ⁶⁸Ga-IMP 288 (0.25 nmol) with a 16 hourinterval. The animal was imaged one hour after the ¹⁸F-FDG and ⁶⁸Ga-IMP288 injection. The panel shows the 3D volume rendering (A), transversesections of the tumor region (B) of the FDG-PET scan, and the 3D volumerendering (C), transverse sections of the tumor region (D) of thepretargeted immunoPET scan.

FIG. 9. Biodistribution of 0.25 nmol Al¹⁸F-IMP 449 (5 MBq) 1 hour afteri.v. injection of 6.0 nmol TF2 16 hours earlier, biodistribution ofAl¹⁸F-B4P 449 without pretargeting, or biodistribution of Al[¹⁸F].Values are given as means±standard deviation.

FIG. 10. Static PET/CT imaging study of a BALB/c nude mouse with asubcutaneous LS174T tumor (0.1 g) on the right side (arrow), thatreceived 6.0 nmol TF2 and 0.25 nmol Al¹⁸F-IMP 449 (5 MBq) intravenouslywith a 16 hour interval. The animal was imaged one hour after injectionof Al¹⁸F-IMP 449. The panel shows the 3D volume rendering (A) posteriorview, and cross sections at the tumor region, (B) coronal, (C) sagittal.

FIG. 11. Structure of IMP 479 (SEQ ID NO:35).

FIG. 12. Structure of IMP 485 (SEQ ID NO:36).

FIG. 13. Structure of IMP 487 (SEQ ID NO:37).

FIG. 14. Synthesis of bis-t-butyl-NODA-MPAA.

FIG. 15. Synthesis of maleimide conjugate of NOTA.

DETAILED DESCRIPTION

The following definitions are provided to facilitate understanding ofthe disclosure herein. Terms that are not explicitly defined are usedaccording to their plain and ordinary meaning.

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

As used herein, “about” means within plus or minus ten percent of anumber. For example, “about 100” would refer to any number between 90and 110.

As used herein, a “peptide” refers to any sequence of naturallyoccurring or non-naturally occurring amino acids of between 2 and 100amino acid residues in length, more preferably between 2 and 10, morepreferably between 2 and 6 amino acids in length. An “amino acid” may bean L-amino acid, a D-amino acid, an amino acid analogue, an amino acidderivative or an amino acid mimetic.

As used herein, the term “pathogen” includes, but is not limited tofungi, viruses, parasites and bacteria, including but not limited tohuman immunodeficiency virus (HIV), herpes virus, cytomegalovirus,rabies virus, influenza virus, hepatitis B virus, Sendai virus, felineleukemia virus, Reovirus, polio virus, human serum parvo-like virus,simian virus 40, respiratory syncytial virus, mouse mammary tumor virus,Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murineleukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Streptococcus agalactiae, Legionella pneumophila, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus, Hemophilus influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, Mycobacterium tuberculosis and Clostridium tetani.

As used herein, a “radiolysis protection agent” refers to any molecule,compound or composition that may be added to an ¹⁸F-labeled complex ormolecule to decrease the rate of breakdown of the ¹⁸F-labeled complex ormolecule by radiolysis. Any known radiolysis protection agent, includingbut not limited to ascorbic acid, may be used.

¹⁸F Labeling Techniques

A variety of techniques for labeling molecules with ¹⁸F are known. Table1 lists the properties of several of the more commonly reportedfluorination procedures. Peptide labeling through carbon often involves¹⁸F-binding to a prosthetic group through nucleophilic substitution,usually in 2- or 3-steps where the prosthetic group is labeled andpurified, attached to the compound, and then purified again. Thisgeneral method has been used to attach prosthetic groups through amidebonds, aldehydes, and “click chemistry” (Marik et al., 2006, BioconjugChem 17:1017-21; Poethko et al., 2004, J Nucl Med 45:892-902; Li et al.,2007, Bioconjug Chem 18:989-93). The most common amide bond-formingreagent has been N-succinimidyl 4-¹⁸F-fluorobenzoate (¹⁸F-SFB), but anumber of other groups have been tested (Marik et al., 2006). In somecases, such as when ¹⁸F-labeled active ester amide-forming groups areused, it may be necessary to protect certain groups on a peptide duringthe coupling reaction, after which they are cleaved. The synthesis ofthis ¹⁸F-SFB reagent and subsequent conjugation to the peptide requiresmany synthetic steps and takes about 2-3 h.

A simpler, more efficient ¹⁸F-peptide labeling method was developed byPoethko et al. (2004), where a 4-¹⁸F-fluorobenzaldehyde reagent wasconjugated to a peptide through an oxime linkage in about 75-90 min,including the dry-down step. The newer “click chemistry” method attaches¹⁸F-labeled molecules onto peptides with an acetylene or azide in thepresence of a copper catalyst (Li et al, 2007; Glaser and Arstad, 2007,Bioconjug Chem 18:989-93). The reaction between the azide and acetylenegroups forms a triazole connection, which is quite stable and forms veryefficiently on peptides without the need for protecting groups. Clickchemistry produces the ¹⁸F-labeled peptides in good yield (−50%) inabout 75-90 min with the dry-down step.

TABLE 1 Summary of selected ¹⁸F-peptide labeling methods. Author/Ref.Schirrmacher Höhne et Li et al. Glaser & Poethko et al. Marik et et al.(2007) al. (2007) Arstad (2004) al (2006) (2008) (2007) AttachmentSilicon Silicon Click Click Aldehyde/oxime Amide Rx steps 2 1 2 2 2 manyRx time 40  115-155 110  65-80 75-90 min 110⁺ (min)^(a) (estimated)(estimated) Yield^(b) 55% 13% 54% 50% 40% 10% HPLC- 1 1 2 1 + 1  2purification distillation steps Specific 225-680 62  high high high highActivity (GBq/μmol) ^(a)Including dry-down time ^(b)Decay corrected

A more recent method of binding ¹⁸F to silicon uses isotopic exchange todisplace ¹⁹F with ¹⁸F (Shirrmacher et al., 2007). Performed at roomtemperature in 10 min, this reaction produces the ¹⁸F-prostheticaldehyde group with high specific activity (225-680 GBOimol;6,100-18,400 Ci/mmol). The ¹⁸F-labeled aldehyde is subsequentlyconjugated to a peptide and purified by HPLC, and the purified labeledpeptide is obtained within 40 min (including dry-down) with ˜55% yield.This was modified subsequently to a single-step process by incorporatingthe silicon into the peptide before the labeling reaction (Hohne et al,2008). However, biodistribution studies in mice with an¹⁸F-silicon-bombesin derivative showed bone uptake increasing over time(1.35±0.47% injected dose (ID)/g at 0.5 h vs. 5.14±2.71% ID/g at 4.0 h),suggesting a release of ¹⁸F from the peptide, since unbound ¹⁸F is knownto localize in bone (Hohne et al., 2008). HPLC analysis of urine showeda substantial amount of ¹⁸F activity in the void volume, whichpresumably is due to ¹⁸F fluoride anion released from the peptide. Itwould therefore appear that the ¹⁸F-silicon labeled molecule was notstable in serum. Substantial hepatobiliary excretion was also reported,attributed to the lipophilic nature of the ¹⁸F-silicon-bindingsubstrate, and requiring future derivatives to be more hydrophilic.Methods of attaching ¹⁸F to boron also have been explored; however, thecurrent process produces conjugates with low specific activity (Ting etal., 2008).

Antibodies and peptides are coupled routinely with radiometals,typically in 15 min and in quantitative yields (Meares et al., 1984, AccChem Res 17:202-209; Scheinberg et al., 1982, Science 215:1511-13). ForPET imaging, ⁶⁴Cu and ⁶⁸Ga have been bound to peptides via a chelate,and have shown reasonably good PET-imaging properties (Heppler et al.,2000, Current Med Chem 7:971-94). Since fluoride binds to most metals,we sought to determine if an ¹⁸F-metal complex could be bound to achelator on a targeting molecule (Tewson, 1989, Nucl Med Biol.16:533-51; Martin, 1996, Coordination Chem Rev 141:23-32). We havefocused on the binding of an Al¹⁸F complex, since aluminum-fluoride canbe relatively stable in vivo (Li, 2003, Crit. Rev Oral Biol Med14:100-114; Antonny et al., 1992, J Biol Chem 267:6710-18). Initialstudies showed the feasibility of this approach to prepare an¹⁸F-labeled peptide for in vivo targeting of cancer with a bispecificantibody (bsMAb) pretargeting system, a highly sensitive and specifictechnique for localizing cancer, in some cases better than ¹⁸F-FDG(fluorodeoxyglucose) (McBride et al., 2008, J Nucl Med (suppl) 49:97 P;Wagner, 2008, J Nucl Med 49:23 N-24N; Karacay et al., 2000, Bioconj Chem11:842-54; Sharkey et al., 2008, Cancer Res 68; 5282-90; Gold Et al.,2008, Cancer Res 68:4819-26; Sharkey et al., 2005, Nature Med11:1250-55; Sharkey et al., 2005, Clin Cancer Res 11:7109s-7121s;McBride et al., 2006, J Nucl Med 47:1678-88; Sharkey et al., 2008,Radiology 246:497-508). These studies revealed that an Al¹⁸F complexcould bind stably to a 1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA), but the yields were low.

In the Examples below, new labeling conditions and several new chelatingmoieties were examined that enhanced yields from about 10% to about 80%,providing a feasible method for ¹⁸F labeling of peptides and othermolecules of use for PET imaging.

Targetable Constructs

In certain embodiments, the moiety labeled with ¹⁸F or other diagnosticand/or therapeutic agents may comprise a peptide or other targetableconstruct. Labeled peptides (or proteins) may be selected to binddirectly to a targeted cell, tissue, pathogenic organism or other targetfor imaging, detection and/or diagnosis. In other embodiments, labeledpeptides may be selected to bind indirectly, for example using abispecific antibody with one or more binding sites for a targetableconstruct peptide and one or more binding sites for a target antigenassociated with a disease or condition. Bispecific antibodies may beused, for example, in a pretargeting technique wherein the antibody maybe administered first to a subject. Sufficient time may be allowed forthe bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue. The distribution of ¹⁸F-labeled targetable constructs may bedetermined by PET scanning or other known techniques.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic. Aside frompeptides, carbohydrates may also be used.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂(SEQ ID NO: 1), wherein DOTA is1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid and HSG is thehistamine succinyl glycyl group. Alternatively, DOTA may be replaced byNOTA (1,4,7-triaza-cyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity. Exemplary methods of peptidesynthesis are disclosed in the Examples below.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

The skilled artisan will realize that although the majority oftargetable constructs disclosed in the Examples below are peptides,other types of molecules may be used as targetable constructs. Forexample, polymeric molecules, such as polyethylene glycol (PEG) may beeasily derivatized with chelating moieties to bind ¹⁸F—Al. Many examplesof such carrier molecules are known in the art and may be utilized,including but not limited to polymers, nanoparticles, microspheres,liposomes and micelles. For use in pretargeted delivery of ¹⁸F, the onlyrequirement is that the carrier molecule comprise one or more chelatingmoieties for attachment of metal-¹⁸F and one or more hapten moieties tobind to a bispecific or multispecific antibody or other targetingmolecule.

Chelating Moieties

In some embodiments, an ¹⁸F-labeled molecule may comprise one or morehydrophilic chelate moieties, which can bind metal ions and also help toensure rapid in vivo clearance. Chelators may be selected for theirparticular metal-binding properties, and may be readily interchanged.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs. Macrocyclic chelators such asNOTA (1,4,7-triaza-cyclononane-1,4,7-triacetic acid), DOTA, TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) and NETA arealso of use with a variety of metals, that may potentially be used asligands for ¹⁸F conjugation.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers are of interest forstably binding nuclides. Porphyrin chelators may be used with numerousmetal complexes. More than one type of chelator may be conjugated to acarrier to bind multiple metal ions. Chelators such as those disclosedin U.S. Pat. No. 5,753,206, especially thiosemicarbazonylglyoxylcysteine(Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelatorsare advantageously used to bind soft acid cations of Tc, Re, Bi andother transition metals, lanthanides and actinides that are tightlybound to soft base ligands. It can be useful to link more than one typeof chelator to a peptide. Because antibodies to a di-DTPA hapten areknown (Barbet et al., U.S. Pat. No. 5,256,395) and are readily coupledto a targeting antibody to form a bispecific antibody, it is possible touse a peptide hapten with cold diDTPA chelator and another chelator forbinding an ¹⁸F complex, in a pretargeting protocol. One example of sucha peptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH₂ (core peptidedisclosed as SEQ ID NO:2). Other hard acid chelators such as DOTA, TETAand the like can be substituted for the DTPA and/or Tscg-Cys groups, andMAbs specific to them can be produced using analogous techniques tothose used to generate the anti-di-DTPA MAb.

Another useful chelator may comprise a NOTA-type moiety, for example asdisclosed in Chong et al. (J. Med. Chem., 2008, 51:118-25). Chong et al.disclose the production and use of a bifunctional C-NETA ligand, basedupon the NOTA structure, that when complexed with ¹⁷⁷Lu or ^(205/206)Bishowed stability in serum for up to 14 days. The chelators are notlimiting and these and other examples of chelators that are known in theart and/or described in the following Examples may be used in thepractice of the invention.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the targetable construct, e.g., withdifferent chelate ring sizes, to bind preferentially to two differenthard acid or soft acid cations, due to the differing sizes of thecations, the geometries of the chelate rings and the preferred complexion structures of the cations. This will permit two different metals,one or both of which may be attached to ¹⁸F, to be incorporated into atargetable construct for eventual capture by a pretargeted bispecificantibody.

Antibodies

Target Antigens

Targeting antibodies of use may be specific to or selective for avariety of cell surface or disease-associated antigens. Exemplary targetantigens of use for imaging or treating various diseases or conditions,such as a malignant disease, a cardiovascular disease, an infectiousdisease, an inflammatory disease, an autoimmune disease, a metabolicdisease, or a neurological (e.g., neurodegenerative) disease may includecarbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22,CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a,CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7,CXCL12, AFP, CEACAM5, CEACAM6, c-met, B7, ED-B of fibronectin, Factor H,FHL-1, Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible factor(HIF), HM1.24, insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α,IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8,IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A,MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, NCA-95, NCA-90, pancreaticcancer mucin, placental growth factor, p53, PLAGL2, prostatic acidphosphatase, PSA, PRAME, PSMA, PDGF, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2),VEGFR, EGFR, complement factors C3, C3a, C3b, C5a, C5, PLAGL2, and anoncogene product.

In certain embodiments, such as imaging or treating tumors, antibodiesof use may target tumor-associated antigens. These antigenic markers maybe substances produced by a tumor or may be substances which accumulateat a tumor site, on tumor cell surfaces or within tumor cells. Amongsuch tumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer”, in Fleisher ed., “The Clinical Biochemistryof Cancer”, page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744, the Examplessection of each of which is incorporated herein by reference. Reports ontumor associated antigens (TAAs) include Mizukami et al., (2005, NatureMed. 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Renet al. (2005, Ann. Surg. 242:55-63), each incorporated herein byreference with respect to the TAAs identified.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

Another marker of interest is transmembrane activator andCAML-interactor (TACT). See Yu et al. Nat. Immunol. 1:252-256 (2000).Briefly, TACI is a marker for B-cell malignancies (e.g., lymphoma). TACIand B-cell maturation antigen (BCMA) are bound by the tumor necrosisfactor homolog—a proliferation-inducing ligand (APRIL). APRIL stimulatesin vitro proliferation of primary B and T-cells and increases spleenweight due to accumulation of B-cells in vivo. APRIL also competes withTALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA andTACI specifically prevent binding of APRIL and block APRIL-stimulatedproliferation of primary B-cells. BCMA-Fc also inhibits production ofantibodies against keyhole limpet hemocyanin and Pneumovax in mice,indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI arerequired for generation of humoral immunity. Thus, APRIL-TALL-I andBCMA-TACI form a two ligand-two receptor pathway involved in stimulationof B and T-cell function.

Where the disease involves a lymphoma, leukemia or autoimmune disorder,targeted antigens may be selected from the group consisting of CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138,CD154, B7, MUC1, Ia, 11, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-Bfibronectin, an oncogene (e.g., c-met or PLAGL2), an oncogene product,CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4)and TRAIL-R2 (DR5).

Methods for Raising Antibodies

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies

The skilled artisan will realize that the targeting molecules of use forimaging, detection and/or diagnosis may incorporate any antibody orfragment known in the art that has binding specificity for a targetantigen associated with a disease state or condition. Such knownantibodies include, but are not limited to, hRl (anti-IGF-1R, U.S.patent application Ser. No. 12/772,645, filed Mar. 12, 2010) hPAM4(anti-pancreatic cancer mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,662,378, U.S.patent application Ser. No. 12/846,062, filed Jul. 29, 2010), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496)the Examples section of each cited patent or application incorporatedherein by reference.

Candidate anti-HIV antibodies include the anti-envelope antibodydescribed by Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), aswell as the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos etal., Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all incorporatedherein by reference.

Where bispecific antibodies are used, the second MAb may be selectedfrom any anti-hapten antibody known in the art, including but notlimited to h679 (U.S. Pat. No. 7,429,381) and 734 (U.S. Pat. Nos.7,429,381; 7,563,439; 7,666,415; and 7,534,431), the Examples section ofeach of which is incorporated herein by reference.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730,300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.) Such knownantibodies are of use for detection and/or imaging of a variety ofdisease states or conditions (e.g., hMN-14 or TF2 (CEA-expressingcarcinomas), hA20 or TF-4 (lymphoma), hPAM4 or TF-10 (pancreaticcancer), RS7 (lung, breast, ovarian, prostatic cancers), hMN-15 or hMN3(inflammation), anti-gp120 and/or anti-gp41 (HIV), anti-platelet andanti-thrombin (clot imaging), anti-myosin (cardiac necrosis), anti-CXCR4(cancer and inflammatory disease)).

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. An antibody fragment can be prepared byproteolytic hydrolysis of the full length antibody or by expression inE. coli or another host of the DNA coding for the fragment. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

A single chain Fv molecule (scFv) comprises a V_(L) domain and a V_(H)domain. The V_(L) and V_(H) domains associate to form a target bindingsite. These two domains are further covalently linked by a peptidelinker (L). Methods for making scFv molecules and designing suitablepeptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80(1995) and R. E. Bird and B. W. Walker, “Single Chain Antibody VariableRegions,” TIBTECH, Vol 9: 132-137 (1991), incorporated herein byreference.

An scFv library with a large repertoire can be constructed by isolatingV-genes from non-immunized human donors using PCR primers correspondingto all known V_(H), V_(kappa) and V₈₀ gene families. See, e.g., Vaughnet al., Nat. Biotechnol., 14: 309-314 (1996). Following amplification,the V_(kappa) and V_(lambda) pools are combined to form one pool. Thesefragments are ligated into a phagemid vector. The scFv linker is thenligated into the phagemid upstream of the V_(L) fragment. The V_(H) andlinker-V_(L) fragments are amplified and assembled on the J_(H) region.The resulting V_(H)-linker-V_(L) fragments are ligated into a phagemidvector. The phagemid library can be panned for binding to the selectedantigen.

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIES 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001) Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (Cabs) (Maass et al., 2007). Alpacas may beimmunized with known antigens and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). These and otherknown antigen-binding antibody fragments may be utilized in the claimedmethods and compositions.

General Techniques for Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the MAb may be amplified using the primersVK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primer setdescribed by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for VK can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the Vκ and V_(H)expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Bispecific and Multispecific Antibodies

Certain embodiments concern pretargeting methods with bispecificantibodies and hapten-bearing targetable constructs. Numerous methods toproduce bispecific or multispecific antibodies are known, as disclosed,for example, in U.S. Pat. No. 7,405,320, the Examples section of whichis incorporated herein by reference. Bispecific antibodies can beproduced by the quadroma method, which involves the fusion of twodifferent hybridomas, each producing a monoclonal antibody recognizing adifferent antigenic site (Milstein and Cuello, Nature, 1983;305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Other methods include improving theefficiency of generating hybrid hybridomas by gene transfer of distinctselectable markers via retrovirus-derived shuttle vectors intorespective parental hybridomas, which are fused subsequently (DeMonte,et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of ahybridoma cell line with expression plasmids containing the heavy andlight chain genes of a different antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv), as discussedabove. Reduction of the peptide linker length to less than 12 amino acidresidues prevents pairing of V_(H) and V_(L) domains on the same chainand forces pairing of V_(H) and V_(L) domains with complementary domainson other chains, resulting in the formation of functional multimers.Polypeptide chains of V_(H) and V_(L) domains that are joined withlinkers between 3 and 12 amino acid residues form predominantly dimers(termed diabodies). With linkers between 0 and 2 amino acid residues,trimers (termed triabody) and tetramers (termed tetrabody) are favored,but the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of V-domains (V_(H)-linker-V_(L)or V_(L)-linker-V_(H)), in addition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL), discussed inmore detail below, has been utilized to produce combinations ofvirtually any desired antibodies, antibody fragments and other effectormolecules (see, e.g., U.S. Patent Application Publ. Nos. 20060228357;20060228300; 20070086942; 20070140966 and 20070264265, the Examplessection of each incorporated herein by reference). The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies, either as naked antibody moieties or in combination with awide range of other effector molecules such as immunomodulators,enzymes, chemotherapeutic agents, chemokines, cytokines, diagnosticagents, therapeutic agents, radionuclides, imaging agents,anti-angiogenic agents, growth factors, oligonucleotides, hormones,peptides, toxins, pro-apoptotic agents, or a combination thereof. Any ofthe techniques known in the art for making bispecific or multispecificantibodies may be utilized in the practice of the presently claimedmethods.

Dock-and-Lock (DNL)

In preferred embodiments, bispecific or multispecific antibodies orother constructs may be produced using the dock-and-lock technology(see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787and 7,666,400, the Examples section of each incorporated herein byreference). The DNL method exploits specific protein/proteininteractions that occur between the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and the anchoring domain (AD) ofA-kinase anchoring proteins (AKAPs) (Baillie et al., FEBS Letters. 2005;579: 3264. Wong and Scott, Nat. Rev. Mol. Cell. Biol. 2004; 5: 959).PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residues(Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to theR subunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanMkt are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAP as an excellent pair of linker modules for dockingany two entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a stably tethered structurethrough the introduction of cysteine residues into both the DDD and ADat strategic positions to facilitate the formation of disulfide bonds.The general methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNLconstructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other diagnostic ortherapeutic agent is attached to a small delivery molecule (targetableconstruct) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Immunoconjugates

Any of the antibodies, antibody fragments or antibody fusion proteinsdescribed herein may be conjugated to a chelating moiety or othercarrier molecule to form an immunoconjugate. Methods for covalentconjugation of chelating moieties and other functional groups are knownin the art and any such known method may be utilized.

For example, a chelating moiety or carrier can be attached at the hingeregion of a reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

Alternatively, the chelating moiety or carrier can be conjugated via acarbohydrate moiety in the Fc region of the antibody. Methods forconjugating peptides to antibody components via an antibody carbohydratemoiety are well-known to those of skill in the art. See, for example,Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al., Int. J. Cancer46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313, the Examplessection of which is incorporated herein by reference. The general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free aminefunction. This reaction results in an initial Schiff base (imine)linkage, which can be stabilized by reduction to a secondary amine toform the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos.5,443,953 and 6,254,868, the Examples section of which is incorporatedherein by reference. The engineered carbohydrate moiety is used toattach the functional group to the antibody fragment.

Other methods of conjugation of chelating agents to proteins are wellknown in the art (see, e.g., U.S. Pat. No. 7,563,433, the Examplessection of which is incorporated herein by reference). Chelates may bedirectly linked to antibodies or peptides, for example as disclosed inU.S. Pat. No. 4,824,659, incorporated herein in its entirety byreference.

Click Chemistry

An alternative method for attaching chelating moieties, peptides orother functional groups to a targeting molecule involves use of clickchemistry reactions. The click chemistry approach was originallyconceived as a method to rapidly generate complex substances by joiningsmall subunits together in a modular fashion. (See, e.g., Kolb et al.,2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.)Various forms of click chemistry reaction are known in the art, such asthe Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoeet al., 2002, J Organic Chem 67:3057-64), which is often referred to asthe “click reaction.” Other alternatives include cycloaddition reactionssuch as the Diels-Alder, nucleophilic substitution reactions (especiallyto small strained rings like epoxy and aziridine compounds), carbonylchemistry formation of urea compounds and reactions involvingcarbon-carbon double bonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is a 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach chelating moieties toantibodies or other targeting molecules in vitro.

Methods of Administration

In various embodiments, bispecific antibodies and targetable constructsmay be used for imaging normal or diseased tissue and organs (see, e.g.U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094;5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679;5,128,119; 5,101,827; and 4,735,210, each incorporated herein byreference in its Examples section).

The administration of a bispecific antibody (bsAb) and an ¹⁸F-labeledtargetable construct may be conducted by administering the bsAb antibodyat some time prior to administration of the targetable construct. Thedoses and timing of the reagents can be readily devised by a skilledartisan, and are dependent on the specific nature of the reagentsemployed. If a bsAb-F(ab′)₂ derivative is given first, then a waitingtime of 24-72 hr (alternatively 48-96 hours) before administration ofthe targetable construct would be appropriate. If an IgG-Fab′ bsAbconjugate is the primary targeting vector, then a longer waiting periodbefore administration of the targetable construct would be indicated, inthe range of 3-10 days. After sufficient time has passed for the bsAb totarget to the diseased tissue, the ¹⁸F-labeled targetable construct isadministered. Subsequent to administration of the targetable construct,imaging can be performed.

Certain embodiments concern the use of multivalent target bindingproteins which have at least three different target binding sites asdescribed in patent application Ser. No. 60/220,782. Multivalent targetbinding proteins have been made by cross-linking several Fab-likefragments via chemical linkers. See U.S. Pat. Nos. 5,262,524; 5,091,542and Landsdorp et al. Euro. J. Immunol. 16: 679-83 (1986). Multivalenttarget binding proteins also have been made by covalently linkingseveral single chain Fv molecules (scFv) to form a single polypeptide.See U.S. Pat. No. 5,892,020. A multivalent target binding protein whichis basically an aggregate of scFv molecules has been disclosed in U.S.Pat. Nos. 6,025,165 and 5,837,242. A trivalent target binding proteincomprising three scFv molecules has been described in Krott et al.Protein Engineering 10(4): 423-433 (1997).

Alternatively, a technique known as “dock-and-lock” (DNL), described inmore detail below, has been demonstrated for the simple and reproducibleconstruction of a variety of multivalent complexes, including complexescomprising two or more different antibodies or antibody fragments. (See,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400, the Examples section of each of which is incorporated hereinby reference.) Such constructs are also of use for the practice of theclaimed methods and compositions described herein.

A clearing agent may be used which is given between doses of thebispecific antibody (bsAb) and the targetable construct. A clearingagent of novel mechanistic action may be used, namely a glycosylatedanti-idiotypic Fab′ fragment targeted against the disease targetingarm(s) of the bsAb. In one example, anti-CEA (MN-14 Ab)×anti-peptidebsAb is given and allowed to accrete in disease targets to its maximumextent. To clear residual bsAb from circulation, an anti-idiotypic Ab toMN-14, termed WI2, is given, preferably as a glycosylated Fab′ fragment.The clearing agent binds to the bsAb in a monovalent manner, while itsappended glycosyl residues direct the entire complex to the liver, whererapid metabolism takes place. Then the ¹⁸F-labeled targetable constructis given to the subject. The WI2 Ab to the MN-14 arm of the bsAb has ahigh affinity and the clearance mechanism differs from other disclosedmechanisms (see Goodwin et al., ibid), as it does not involvecross-linking, because the WI2-Fab′ is a monovalent moiety. However,alternative methods and compositions for clearing agents are known andany such known clearing agents may be used.

Formulation and Administration

The ¹⁸F-labeled molecules may be formulated to obtain compositions thatinclude one or more pharmaceutically suitable excipients, one or moreadditional ingredients, or some combination of these. These can beaccomplished by known methods to prepare pharmaceutically usefuldosages, whereby the active ingredients (i.e., the ¹⁸F-labeledmolecules) are combined in a mixture with one or more pharmaceuticallysuitable excipients. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell known to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The preferred route for administration of the compositions describedherein is parenteral injection. Injection may be intravenous,intraarterial, intralymphatic, intrathecal, or intracavitary (i.e.,parenterally). In parenteral administration, the compositions will beformulated in a unit dosage injectable form such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives. Other methodsof administration, including oral administration, are also contemplated.

Formulated compositions comprising ¹⁸F-labeled molecules can be used forintravenous administration via, for example, bolus injection orcontinuous infusion. Compositions for injection can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. Compositions can also take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and cancontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the compositions can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The compositions may be administered in solution. The pH of the solutionshould be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. Theformulation thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate, TRIS(hydroxymethyl)aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The formulatedsolution may also contain a salt, such as sodium chloride or potassiumchloride in a concentration of 50 to 150 mM. An effective amount of astabilizing agent such as glycerol, albumin, a globulin, a detergent, agelatin, a protamine or a salt of protamine may also be included. Thecompositions may be administered to a mammal subcutaneously,intravenously, intramuscularly or by other parenteral routes. Moreover,the administration may be by continuous infusion or by single ormultiple boluses.

Where bispecific antibodies are administered, for example in apretargeting technique, the dosage of an administered antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, for imaging purposes it is desirable to provide therecipient with a dosage of bispecific antibody that is in the range offrom about 1 mg to 200 mg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. Typically, it is desirable to provide the recipient with adosage that is in the range of from about 10 mg per square meter of bodysurface area or 17 to 18 mg of the antibody for the typical adult,although a lower or higher dosage also may be administered ascircumstances dictate. Examples of dosages of bispecific antibodies thatmay be administered to a human subject for imaging purposes are 1 to 200mg, more preferably 1 to 70 mg, most preferably 1 to 20 mg, althoughhigher or lower doses may be used.

In general, the dosage of ¹⁸F label to administer will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Preferably, a saturatingdose of the ¹⁸F-labeled molecules is administered to a patient. Foradministration of ¹⁸F-labeled molecules, the dosage may be measured bymillicuries. A typical range for ¹⁸F imaging studies would be five to 10mCi.

Administration of Peptides

Various embodiments of the claimed methods and/or compositions mayconcern one or more ¹⁸F-labeled peptides to be administered to asubject. Administration may occur by any route known in the art,including but not limited to oral, nasal, buccal, inhalational, rectal,vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial, intrathecal or intravenous injection.Where, for example, ¹⁸F-labeled peptides are administered in apretargeting protocol, the peptides would preferably be administeredi.v.

Unmodified peptides administered orally to a subject can be degraded inthe digestive tract and depending on sequence and structure may exhibitpoor absorption across the intestinal lining. However, methods forchemically modifying peptides to render them less susceptible todegradation by endogenous proteases or more absorbable through thealimentary tract are well known (see, for example, Blondelle et al.,1995, Biophys. J. 69:604-11; Ecker and Crooke, 1995, Biotechnology13:351-69; Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMISTRY AND DRUGDISCOVERY, VOL. I, ed. Wollf, John Wiley & Sons; Goodman and Shao, 1996,Pure & Appl. Chem. 68:1303-08). Methods for preparing libraries ofpeptide analogs, such as peptides containing D-amino acids;peptidomimetics consisting of organic molecules that mimic the structureof a peptide; or peptoids such as vinylogous peptoids, have also beendescribed and may be used to construct peptide based ¹⁸F-labeledmolecules suitable for oral administration to a subject.

In certain embodiments, the standard peptide bond linkage may bereplaced by one or more alternative linking groups, such as CH₂—NH,CH₂—S, CH₂—CH₂, CH═CH, CO—CH₂, CHOH—CH₂ and the like. Methods forpreparing peptide mimetics are well known (for example, Hruby, 1982,Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest etal., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int. J. Pept.Res. 14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Pat.Nos. 5,169,862; 5,539,085; 5,576,423, 5,051,448, 5,559,103.) Peptidemimetics may exhibit enhanced stability and/or absorption in vivocompared to their peptide analogs.

Alternatively, peptides may be administered by oral delivery usingN-terminal and/or C-terminal capping to prevent exopeptidase activity.For example, the C-terminus may be capped using amide peptides and theN-terminus may be capped by acetylation of the peptide. Peptides mayalso be cyclized to block exopeptidases, for example by formation ofcyclic amides, disulfides, ethers, sulfides and the like.

Peptide stabilization may also occur by substitution of D-amino acidsfor naturally occurring L-amino acids, particularly at locations whereendopeptidases are known to act. Endopeptidase binding and cleavagesequences are known in the art and methods for making and using peptidesincorporating D-amino acids have been described (e.g., U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Jun. 14,2004, the Examples section of which is incorporated herein byreference). In certain embodiments, peptides and/or proteins may beorally administered by co-formulation with proteinase- and/orpeptidase-inhibitors.

Other methods for oral delivery of therapeutic peptides are disclosed inMehta (“Oral delivery and recombinant production of peptide hormones,”June 2004, BioPharm International). The peptides are administered in anenteric-coated solid dosage form with excipients that modulateintestinal proteolytic activity and enhance peptide transport across theintestinal wall. Relative bioavailability of intact peptides using thistechnique ranged from 1% to 10% of the administered dosage. Insulin hasbeen successfully administered in dogs using enteric-coatedmicrocapsules with sodium cholate and a protease inhibitor (Ziv et al.,1994, J. Bone Miner. Res. 18 (Suppl. 2):792-94. Oral administration ofpeptides has been performed using acylcarnitine as a permeation enhancerand an enteric coating (Eudragit L30D-55, Rohm Pharma Polymers, seeMehta, 2004). Excipients of use for orally administered peptides maygenerally include one or more inhibitors of intestinalproteases/peptidases along with detergents or other agents to improvesolubility or absorption of the peptide, which may be packaged within anenteric-coated capsule or tablet (Mehta, 2004). Organic acids may beincluded in the capsule to acidify the intestine and inhibit intestinalprotease activity once the capsule dissolves in the intestine (Mehta,2004). Another alternative for oral delivery of peptides would includeconjugation to polyethylene glycol (PEG)-based amphiphilic oligomers,increasing absorption and resistance to enzymatic degradation (Solteroand Ekwuribe, 2001, Pharm. Technol. 6:110).

Imaging Using Labeled Molecules

Methods of imaging using labeled molecules are well known in the art,and any such known methods may be used with the ¹⁸F-labeled moleculesdisclosed herein. See, e.g., U.S. Pat. Nos. 6,241,964; 6,358,489;6,953,567 and published U.S. Patent Application Publ. Nos. 20050003403;20040018557; 20060140936, the Examples section of each incorporatedherein by reference. See also, Page et al., Nuclear Medicine AndBiology, 21:911-919, 1994; Choi et al., Cancer Research 55:5323-5329,1995; Zalutsky et al., J. Nuclear Med., 33:575-582, 1992; Woessner et.al. Magn. Reson. Med. 2005, 53: 790-99.

In certain embodiments, ¹⁸F-labeled molecules may be of use in imagingnormal or diseased tissue and organs, for example using the methodsdescribed in U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095;5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902;5,328,679; 5,128,119; 5,101,827; and 4,735,210, each incorporated hereinby reference. Such imaging can be conducted by direct ¹⁸F labeling ofthe appropriate targeting molecules, or by a pretargeted imaging method,as described in Goldenberg et al. (2007, Update Cancer Ther. 2:19-31);Sharkey et al. (2008, Radiology 246:497-507); Goldenberg et al. (2008,J. Nucl. Med. 49:158-63); Sharkey et al. (2007, Clin. Cancer Res.13:5777s-5585s); McBride et al. (2006, J. Nucl. Med. 47:1678-88);Goldenberg et al. (2006, J. Clin. Oncol. 24:823-85), see also U.S.Patent Publication Nos. 20050002945, 20040018557, 20030148409 and20050014207, each incorporated herein by reference.

Methods of diagnostic imaging with labeled peptides or MAbs arewell-known. For example, in the technique of immunoscintigraphy, ligandsor antibodies are labeled with a gamma-emitting radioisotope andintroduced into a patient. A gamma camera is used to detect the locationand distribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Alsopreferred is the use of positron-emitting radionuclides (PET isotopes),such as with an energy of 511 keV, such as ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, and ¹²⁴I.Such radionuclides may be imaged by well-known PET scanning techniques.

In preferred embodiments, the ¹⁸F-labeled peptides, proteins and/orantibodies are of use for imaging of cancer. Examples of cancersinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, colorectal cancer, endometrial cancer or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penilecarcinoma, as well as head and neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma,Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult PrimaryLiver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma,AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer,Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, BreastCancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System(Primary) Lymphoma, Central Nervous System Lymphoma, CerebellarAstrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, ChildhoodBrain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood CerebralAstrocytoma, Childhood Extracranial Germ Cell Tumors, ChildhoodHodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamicand Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, ChildhoodMedulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal andSupratentorial Primitive Neuroectodermal Tumors, Childhood Primary LiverCancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,Childhood Visual Pathway and Hypothalamic Glioma, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-CellLymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma andRelated Tumors, Exocrine Pancreatic Cancer, Extracranial Germ CellTumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, EyeCancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer,Gastric Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalTumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin'sDisease, Hodgkin's Lymphoma, Hypergammaglobulinemia, HypopharyngealCancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, LaryngealCancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, PenileCancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used todetect or diagnose malignant or premalignant conditions. Such uses areindicated in conditions known or suspected of preceding progression toneoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia,pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be detected include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be detected include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps,colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL) and anti-CD74 (e.g., hLL1) antibodies can be used totarget atherosclerotic plaques. Abciximab (anti-glycoprotein IIb/IIIa)has been approved for adjuvant use for prevention of restenosis inpercutaneous coronary interventions and the treatment of unstable angina(Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3 antibodies havebeen reported to reduce development and progression of atherosclerosis(Steffens et al., 2006, Circulation 114:1977-84). Treatment withblocking MIF antibody has been reported to induce regression ofestablished atherosclerotic lesions (Sanchez-Madrid and Sessa, 2010,Cardiovasc Res 86:171-73). Antibodies against oxidized LDL also induceda regression of established atherosclerosis in a mouse model (Ginsberg,2007, J Am Coll Cardiol 52:2319-21). Anti-ICAM-1 antibody was shown toreduce ischemic cell damage after cerebral artery occlusion in rats(Zhang et al., 1994, Neurology 44:1747-51). Commercially availablemonoclonal antibodies to leukocyte antigens are represented by: OKTanti-T-cell monoclonal antibodies (available from Ortho PharmaceuticalCompany) which bind to normal T-lymphocytes; the monoclonal antibodiesproduced by the hybridomas having the ATCC accession numbers HB44, HB55,HB12, HB78 and HB2; G7E11, W8E7, NKP15 and GO22 (Becton Dickinson);NEN9.4 (New England Nuclear); and FMC11 (Sera Labs). A description ofantibodies against fibrin and platelet antigens is contained in Knight,Semin. Nucl. Med., 20:52-67 (1990).

In one embodiment, a pharmaceutical composition of the present inventionmay be used to treat a subject having a metabolic disease, suchamyloidosis, or a neurodegenerative disease, such as Alzheimer'sdisease, amyotrophic lateral sclerosis (ALS), Parkinson's disease,Huntington's disease, olivopontocerebellar atrophy, multiple systematrophy, progressive supranuclear palsy, diffuse lewy body disease,corticodentatonigral degeneration, progressive familial myoclonicepilepsy, strionigral degeneration, torsion dystonia, familial tremor,Gilles de la Tourette syndrome or Hallervorden-Spatz disease.Bapineuzumab is in clinical trials for Alzheimer's disease therapy.Other antibodies proposed for therapy of Alzheimer's disease include Alz50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition. Antibodies againstmutant SOD1, produced by hybridoma cell lines deposited with theInternational Depositary Authority of Canada (accession Nos.ADI-290806-01, ADI-290806-02, ADI-290806-03) have been proposed fortherapy of ALS, Parkinson's disease and Alzheimer's disease (see U.S.Patent Appl. Publ. No. 20090068194). Anti-CD3 antibodies have beenproposed for therapy of type 1 diabetes (Cernea et al., 2010, DiabetesMetab Rev 26:602-05). In addition, a pharmaceutical composition of thepresent invention may be used to treat a subject having animmune-dysregulatory disorder, such as graft-versus-host disease ororgan transplant rejection.

The exemplary conditions listed above that may be detected, diagnosedand/or imaged are not limiting. The skilled artisan will be aware thatantibodies, antibody fragments or targeting peptides are known for awide variety of conditions, such as autoimmune disease, cardiovasculardisease, neurodegenerative disease, metabolic disease, cancer,infectious disease and hyperproliferative disease. Any such conditionfor which an ¹⁸F-labeled molecule, such as a protein or peptide, may beprepared and utilized by the methods described herein, may be imaged,diagnosed and/or detected as described herein.

Kits

Various embodiments may concern kits containing components suitable forimaging, diagnosing and/or detecting diseased tissue in a patient usinglabeled compounds. Exemplary kits may contain an antibody, fragment orfusion protein, such as a bispecific antibody of use in pretargetingmethods as described herein. Other components may include a targetableconstruct for use with such bispecific antibodies. In preferredembodiments, the targetable construct is pre-conjugated to a chelatinggroup that may be used to attach an Al¹⁸F complex or a complex of ¹⁸Fwith a different metal. However, in alternative embodiments it iscontemplated that a targetable construct may be attached to one or moredifferent diagnostic agents, such as ⁶⁸Ga.

If the composition containing components for administration is notformulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused for certain applications.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES Example 1 ¹⁸F Labeling of Peptide IMP 272

The first peptide that was prepared and ¹⁸F-labeled was IMP 272:

DTPA-Gln-Ala-Lys (HS G)-D-Tyr-Lys (HSG)-NH₂ (SEQ ID NO:3)

Acetate buffer solution—Acetic acid, 1.509 g was diluted in ˜160 mLwater and the pH was adjusted by the addition of 1M NaOH then diluted to250 mL to make a 0.1M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1M NaOAcsolution at pH 4 to provide a 2 mM A1 stock solution.

IMP 272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP 272was dissolved in 364 μL of the 0.1M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

F-18 Labeling of IMP 272—A 3 μL aliquot of the aluminum stock solutionwas placed in a REACTI-VIALT™ and mixed with 50 μL ¹⁸F (as received) and3 μL of the IMP 272 solution. The solution was heated in a heating blockat 110° C. for 15 min and analyzed by reverse phase HPLC. The HPLC trace(not shown) showed 93% free ¹⁸F and 7% bound to the peptide. Anadditional 10 μL of the IMP 272 solution was added to the reaction andit was heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% ¹⁸F at the void volume and 92% of the activityattached to the peptide. The remainder of the peptide solution wasincubated at room temperature with 150 μL PBS for ˜1 hr and thenexamined by reverse phase HPLC. The HPLC (not shown) showed 58% ¹⁸Funbound and 42% still attached to the peptide. The data indicate that¹⁸F—Al-DTPA complex may be unstable when mixed with phosphate.

Example 2 IMP 272 ¹⁸F Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10⁹ mol) was placed in apolypropylene cone vial and mixed with 75 μL ¹⁸F (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁻⁸ mol) IMP 272 solution in 0.1M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown). These results demonstrate that the ¹⁸F metal labeling techniqueis not limited to an aluminum ligand, but can also utilize other metalsas well. With different metal ligands, different chelating moieties maybe utilized to optimize binding of an F-18-metal conjugate.

Example 3 Production and Use of a Serum-Stable ¹⁸F-Labeled Peptide IMP449

IMP 449

NOTA-ITC benzyl-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:4)

The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:5)was made on Sieber Amide resin by adding the following amino acids tothe resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, theAloc was cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make thedesired peptide. The peptide was then cleaved from the resin andpurified by HPLC to produce IMP 448, which was then coupled toITC-benzyl NOTA.

IMP 448 (0.0757 g, 7.5×10⁻⁵ mol) was mixed with 0.0509 g (9.09×10⁵ mol)ITC benzyl NOTA and dissolved in 1 mL water. Potassium carbonateanhydrous (0.2171 g) was then slowly added to the stirred peptide/NOTAsolution. The reaction solution was pH 10.6 after the addition of allthe carbonate. The reaction was allowed to stir at room temperatureovernight. The reaction was carefully quenched with 1M HCl after 14 hrand purified by HPLC to obtain 48 mg of IMP 449.

¹⁸F Labeling of IMP 449

IMP 449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2 mM peptidesolution) 0.1M NaOAc pH 4.02. Three microliters of a 2 mM solution of Alin a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of ¹⁸F. Thesolution was then mixed with 20 μL of the 2 mM IMP 449 solution andheated at 105° C. for 15 min. Reverse Phase HPLC analysis showed 35%(t_(R) 10 min) of the activity was attached to the peptide and 65% ofthe activity was eluted at the void volume of the column (3.1 min, notshown) indicating that the majority of activity was not associated withthe peptide. The crude labeled mixture (5 μL) was mixed with pooledhuman serum and incubated at 37° C. An aliquot was removed after 15 minand analyzed by HPLC. The HPLC showed 9.8% of the activity was stillattached to the peptide (down from 35%). Another aliquot was removedafter 1 hr and analyzed by HPLC. The HPLC showed 7.6% of the activitywas still attached to the peptide (down from 35%), which was essentiallythe same as the 15 min trace (data not shown).

High Dose ¹⁸F Labeling

Further studies with purified IMP 449 demonstrated that the ¹⁸F-labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. Additional studies wereperformed in which the IMP 449 was prepared in the presence of ascorbicacid as a stabilizing agent. In those studies (not shown), themetal-¹⁸F-peptide complex showed no detectable decomposition in serumafter 4 hr at 37° C. The mouse urine 30 min after injection of¹⁸F-labeled peptide was found to contain ¹⁸F bound to the peptide (notshown). These results demonstrate that the ¹⁸F-labeled peptidesdisclosed herein exhibit sufficient stability under approximated in vivoconditions to be used for ¹⁸F imaging studies.

Since IMP 449 peptide contains a thiourea linkage, which is sensitive toradiolysis, several products are observed by RP-HPLC. However, whenascorbic acid is added to the reaction mixture, the side productsgenerated are markedly reduced.

Example 4 Preparation of DNL Constructs for ¹⁸F Imaging by Pretargeting

The DNL technique may be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibodies or fragments thereofor other effector moieties. For certain preferred embodiments, IgGantibodies, Fab fragments or other proteins or peptides may be producedas fusion proteins containing either a DDD (dimerization and dockingdomain) or AD (anchoring domain) sequence. Bispecific antibodies may beformed by combining a Fab-DDD fusion protein of a first antibody with aFab-AD fusion protein of a second antibody. Alternatively, constructsmay be made that combine IgG-AD fusion proteins with Fab-DDD fusionproteins. For purposes of ¹⁸F detection, an antibody or fragmentcontaining a binding site for an antigen associated with a target tissueto be imaged, such as a tumor, may be combined with a second antibody orfragment that binds a hapten on a targetable construct, such as IMP 449,to which a metal-¹⁸F can be attached. The bispecific antibody (DNLconstruct) is administered to a subject, circulating antibody is allowedto clear from the blood and localize to target tissue, and the¹⁸F-labeled targetable construct is added and binds to the localizedantibody for imaging.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD₂-fusion protein module can be combined with any correspondingAD-fusion protein module to generate a bispecific DNL construct. Fordifferent types of constructs, different AD or DDD sequences may beutilized. The following DDD sequences are based on the DDD moiety of PKAMkt, while the AD sequences are based on the AD moiety of the optimizedsynthetic AKAP-IS sequence (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445).

DDD1: (SEQ ID NO: 6) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2:(SEQ ID NO: 7) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1:(SEQ ID NO: 8) QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 9)CGQIEYLAKQIVDNAIQQAGC

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1). To generate Fab-AD expression vectors, the sequences for thehinge, CH₂ and CH3 domains of IgG are replaced with a sequence encodingthe first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17residue synthetic AD called AKAP-IS (referred to as AD1), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge followed by four glycines and aserine, with the final two codons (GS) comprising a Bam HI restrictionsite. The 410 bp PCR amplimer was cloned into the pGemT PCR cloningvector (Promega, Inc.) and clones were screened for inserts in the T7(5′) orientation.

A duplex oligonucleotide was synthesized by to code for the amino acidsequence of DDD1 preceded by 11 residues of a linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below, with the DDD1 sequence underlined.

(SEQ ID NO: 10) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFT RLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, thatoverlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174by DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase. Following primerextension, the duplex was amplified by PCR. The amplimer was cloned intopGemT and screened for inserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below, with the sequence of AD1underlined.

(SEQ ID NO: 11) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the pGemT vector and screened for inserts in the T7 (5′)orientation.

Ligating DDD1 with CH1

A 190 by fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

Ligating AD1 with CH1

A 110 by fragment containing the AD1 sequence was excised from pGemTwith BamHI and NotI and then ligated into the same sites in CH1-pGemT togenerate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-based vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/Eagifragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was convertedto C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restrictionendonucleases to remove the CH1-CH3 domains and insertion of theCH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hRl, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

Two overlapping, complimentary oligonucleotides, which comprise thecoding sequence for part of the linker peptide and residues 1-13 ofDDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, whichwas prepared by digestion with BamHI and Psll, to generate the shuttlevector CH1-DDD2-pGemT. A 507 by fragment was excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vectorhMN14(I)-pdHL2, which was prepared by digestion with Sad and EagI. Thefinal expression construct was designated C-DDD2-Fd-hMN-14-pdHL2.Similar techniques have been utilized to generated DDD2-fusion proteinsof the Fab fragments of a number of different humanized antibodies.

H679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchor domain sequence of AD2 appendedto the carboxyl terminal end of the CH1 domain via a 14 amino acidresidue Gly/Ser peptide linker. AD2 has one cysteine residue precedingand another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-pGemT, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 5 Generation of TF2 DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). The additional bands suggest that disulfideformation is incomplete under the experimental conditions (not shown).Reducing SDS-PAGE shows that any additional bands apparent in thenon-reducing gel are product-related (not shown), as only bandsrepresenting the constituent polypeptides of TF2 are evident. MALDI-TOFmass spectrometry (not shown) revealed a single peak of 156,434 Da,which is within 99.5% of the calculated mass (157,319 Da) of TF2.

The functionality of TF2 was determined by BIACORE assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 6 Production of TF10 DNL Construct

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The TF10 bispecific ([hPAM4]₂×h679) antibody was producedusing the method disclosed for production of the (anti CEA)₂×anti HSGbsAb TF2, as described above. The TF10 construct bears two humanizedPAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD2. The reaction mixture was incubated at room temperaturefor 24 hours under mild reducing conditions using 1 mM reducedglutathione. Following reduction, the DNL reaction was completed by mildoxidation using 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

Example 7 Sequence Variants for DNL

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the cytokine-MAb DNL complex comprise the amino acid sequences ofAD1 or AD2 and DDD1 or DDD2, as discussed above. However, in alternativeembodiments sequence variants of AD and/or DDD moieties may be utilizedin construction of the DNL complexes. For example, there are only fourvariants of human PKA DDD sequences, corresponding to the DDD moietiesof PKA RIα, RIIα, RIβ and RIIβ. The RIIα DDD sequence is the basis ofDDD1 and DDD2 disclosed above. The four human PKA DDD sequences areshown below. The DDD sequence represents residues 1-44 of RIIα, 1-44 ofRIIβ, 12-61 of RIα and 13-66 of RIβ. (Note that the sequence of DDD1 ismodified slightly from the human PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 12)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ(SEQ ID NO: 13) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQ ILAPKA RIIα (SEQ ID NO: 14) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 15) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:6below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

(SEQ ID NO: 6) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:8), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:8. The skilledartisan will realize that in designing sequence variants of the ADsequence, one would desirably avoid changing any of the underlinedresidues, while conservative amino acid substitutions might be made forresidues that are less critical for DDD binding.

AKAP-IS sequence (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA

Gold (2006) utilized crystallography and peptide screening to develop aSuperAKAP-IS sequence (SEQ ID NO:16), exhibiting a five order ofmagnitude higher selectivity for the RII isoform of PKA compared withthe R1 isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, which increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:17-19. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:9,the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 16) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 17) QIEYKAKQIVDHAIHQA(SEQ ID NO: 18) QIEYHAKQIVDHAIHQA (SEQ ID NO: 19) QIEYVAKQIVDHAIHQA

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:20-22. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:20), RIAD (SEQ ID NO:21) and PV-38 (SEQ IDNO:22). The Ht-31 peptide exhibited a greater affinity for the RHisoform of PKA, while the RIAD and PV-38 showed higher affinity for R1.

Ht31 (SEQ ID NO: 20) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 21)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 22) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:8). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:23-25.

AKAP-IS (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA AKAP7δ-wt-pep (SEQ ID NO: 23)PEDAELVRLSKRLVENAVLKAVQQY AKAP7δ-L304T-pep (SEQ ID NO: 24)PEDAELVRTSKRLVENAVLKAVQQY AKAP7δ-L308D-pep (SEQ ID NO: 25)PEDAELVRLSKRDVENAVLKAVQQY

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:6. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. The skilled artisan will realize that indesigning sequence variants of DDD, it would be most preferred to avoidchanging the most conserved residues (italicized), and it would bepreferred to also avoid changing the conserved residues (underlined),while conservative amino acid substitutions may be considered forresidues that are neither underlined nor italicized.

(SEQ ID NO: 6) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

Example 8 In Vivo Studies with Pretargeting Antibody and ¹⁸F-LabeledPeptide

¹⁸F-labeled IMP 449 was prepared as follows. The ¹⁸F, 54.7 mCi in ˜0.5mL was mixed with 3 μL 2 mM Al in 0.1M NaOAc pH 4 buffer. After 3 min 10μL of 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer was added and thereaction was heated in a 96° C. heating block for 15 min. The crudelabeled peptide was then purified by HPLC on a C₁₈ column. The HPLCpurified peptide sample was further processed by diluting the fractionsof interest two fold in water and placing the solution in the barrel ofa 1 cc WATERS® HLB column. The cartridge was eluted with 3×1 mL water toremove acetonitrile and TFA followed by 400 μL 1:1 EtOH/H₂O to elute the¹⁸F-labeled peptide. The purified [Al¹⁸F] IMP 449 eluted as a singlepeak on an analytical HPLC C₁₈ column (not shown).

Taconic nude mice bearing the four slow-growing sc CaPanl xenograftswere used for in vivo studies. Three of the mice were injected with TF10(162 μg) followed with [Al¹⁸F] IMP 449 18 h later. TF10 is a humanizedbispecific antibody of use for tumor imaging studies, with divalentbinding to the PAM-4 defined tumor antigen and monovalent binding to HSG(see, e.g., Gold et al., 2007, J. Clin. Oncol. 25(18S):4564). One mousewas injected with peptide alone. All of the mice were necropsied at 1 hpost peptide injection. Tissues were counted immediately. Comparison ofmean distributions showed substantially higher levels of ¹⁸F-labeledpeptide localized in the tumor than in any normal tissues in thepresence of tumor-targeting bispecific antibody.

Tissue uptake was similar in animals given the [Al¹⁸F] IMP 449 alone orin a pretargeting setting (Table 2). Uptake in the human pancreaticcancer xenograft, CaPanl, at 1 h was increased 5-fold in the pretargetedanimals as compared to the peptide alone (4.6±0.9% ID/g vs. 0.89% ID/g).Exceptional tumor/nontumor ratios were achieved at this time (e.g.,tumor/blood and liver ratios were 23.4±2.0 and 23.5±2.8, respectively).

TABLE 2 Tissue uptake at 1 h post peptide injection, mean and theindividual animals: TF10 (162 μg) -→18 h → [Al¹⁸F] IMP 449 [Al¹⁸F] IMP449 (10:1) alone Tissue n Mean SD Animal 1 Animal 2 Animal 3 Animal 1Tumor 3 4.591 0.854 4.330 5.546 3.898 0.893 (mass) (0.675 g) (0.306 g)(0.353 g) (0.721 g) Liver 3 0.197 0.041 0.163 0.242 0.186 0.253 Spleen 30.202 0.022 0.180 0.224 0.200 0.226 Kidney 3 5.624 0.531 5.513 6.2025.158 5.744 Lung 3 0.421 0.197 0.352 0.643 0.268 0.474 Blood 3 0.1960.028 0.204 0.219 0.165 0.360 Stomach 3 0.123 0.046 0.080 0.172 0.1180.329 Small Int. 3 0.248 0.042 0.218 0.295 0.230 0.392 Large Int. 30.141 0.094 0.065 0.247 0.112 0.113 Pancreas 3 0.185 0.078 0.259 0.1940.103 0.174 Spine 3 0.394 0.427 0.140 0.888 0.155 0.239 Femur 3 3.8994.098 2.577 8.494 0.625 0.237 Brain 3 0.064 0.041 0.020 0.072 0.1000.075 Muscle 3 0.696 0.761 0.077 1.545 0.465 0.162

Example 9 Biodistribution of [Al¹⁸F] IMP 449 With Pretargeting Antibody

The goal of the study was to examine biodistribution of [Al¹⁸F] IMP 449in nude mice bearing sc LS174 T xenografts after pretargeting withbispecific antibody TF2.

[Al¹⁸F] IMP 449: Labeling was performed as described above except the¹⁸F was purified on a QMA cartridge before labeling as described by Kimet. al. (Applied Radiation and Isotopes 61, 2004, 1241-46). The columnwas eluted with 1 mL 0.4 M KHCO₃ in 200 μL fractions. The pH of the ¹⁸Fsolution was adjusted with 10 μL of glacial acetic acid and ¹⁸F from thepeak fraction was mixed with 3 of 2 mM Al in 0.1M pH 4 NaOAc buffer. Thesample was then mixed with 10 μL of 0.05 M IMP 449 in 0.5 M NaOAc bufferat pH 4 and the reaction solution was heated at 94° C. for 15 mM. The[Al¹⁸F] IMP 449 was purified by RP-HPLC. The fraction containing theproduct was put through an HLB column to exchange the buffer. Theproduct was eluted with 400 μL 1:1 EtOH:H₂O, Since the yield was low,the specific activity was low and more peptide was injected into mice,resulting in a bsMAb:peptide ratio of 6.9:1 instead of 10:1.

Results

The biodistribution of pretargeted ¹⁸F labeled IMP 449 in mice issummarized in Table 3. The labeled peptide showed a high level oflocalization to tumor tissues in the presence of the bispecific TF2antibody. In the absence of TF2, the labeled peptide did not showsignificantly higher localization to tumor than to normal tissues (notshown).

TABLE 3 Mice were injected with TF2 (163.2 μg, 1.035 × 10⁻⁹ mol) i.v.followed by [Al¹⁸F] IMP 449 (1.5 × 10⁻¹⁰ mol) 16 h later. Peptide tissueuptake (% ID/g) at 1 h post peptide injection is shown below. Tissue nMean SD Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 Tumor 5 7.624 3.0805.298 7.848 12.719 5.118 7.136 Liver 5 0.172 0.033 0.208 0.143 0.1960.131 0.180 Spleen 5 0.142 0.059 0.239 0.081 0.132 0.118 0.140 Kidney 52.191 0.125 2.313 2.141 2.154 2.319 2.027 Lung 5 0.315 0.094 0.474 0.2300.300 0.305 0.265 Blood 5 0.269 0.143 0.431 0.395 0.132 0.126 0.260Stomach 5 0.218 0.341 0.827 0.041 0.098 0.054 0.070 Small Int. 5 0.3510.313 0.903 0.185 0.297 0.170 0.198 Large Int. 5 0.069 0.028 0.076 0.0430.111 0.073 0.042 Femur 5 0.625 0.358 0.869 0.146 0.811 0.957 0.344Spine 5 0.585 0.569 0.159 0.119 0.493 1.526 0.626 Brain 5 0.029 0.0050.033 0.021 0.035 0.026 0.028 Muscle 5 0.736 0.970 0.190 0.064 0.4942.438 0.496 Body Wt. 5 24.69 1.20 23.05 26.36 24.45 24.48 25.11

This study shows that the simple, reproducible methods and compositionsdescribed herein produce ¹⁸F-labeled targeting peptides suitable for usein in vivo imaging of a variety of disease states. The skilled artisanwill realize that the bispecific antibodies disclosed above are notlimiting, but may comprise any known antibodies against a wide varietyof disease or pathogen target antigens. Nor is the method limited topretargeting with bispecific antibodies. In other embodiments, moleculesor complexes that directly bind to target cells, tissues or organisms tobe imaged may be labeled with ¹⁸F using the methods disclosed herein andadministered to a subject for PET imaging (see Examples below).

The Al¹⁸F-labeled peptides, exemplified by IMP 449, are sufficientlystable under in vivo conditions to be utilized in known imagingprotocols, such as PET scanning. Further, the claimed methods result inpreparation of ¹⁸F-labeled targeting peptides that are ready forinjection within 1 hour of preparation time, well within the decay timeof ¹⁸F to allow suitable imaging procedures to be performed. Finally,the described and claimed methods result in minimal exposure of theoperator to radioisotope exposure, compared with known methods ofpreparing ¹⁸F-labeled compounds for imaging studies.

Example 10 In Vivo Imaging Using ¹⁸F-Labeled Peptides and Comparisonwith ¹⁸F[FDG]

In vivo imaging techniques using pretargeting with bispecific antibodiesand labeled targeting peptides were used to successfully detect tumorsof relatively small size. The ¹⁸F was purified on a WATERS® ACCELL™ PlusQMA Light cartridge. The ¹⁸F eluted with 0.4 M KHCO₃ was mixed with 3 μL2 mM Al³⁺ in a pH 4 acetate buffer. The Al¹⁸F solution was then injectedinto the ascorbic acid IMP 449 labeling vial and heated to 105° C. for15 min. The reaction solution was cooled and mixed with 0.8 mL DI water.The reaction contents were loaded on a WATERS® OASIS® icc HLB Column andeluted with 2×200 μL 1:1 EtOH/H₂O. TF2 was prepared as described above.TF2 binds divalently to carcinoembryonic antigen (CEA) and monovalentlyto the synthetic hapten, HSG (histamine-succinyl-glycine).

Biodistribution and MicroPET Imaging.

Six-week-old NCr nu-m female nude mice were implanted s.c. with thehuman colonic cancer cell line, LS174T (ATCC, Manassas, Va.). Whentumors were visibly established, pretargeted animals were injectedintravenously with 162 μg (˜1 nmole/0.1 mL) TF2 or TF10 (controlnon-targeting tri-Fab bsMAb), and then 16-18 h later, ˜0.1 nmol of[Al¹⁸F] IMP 449 (84 μCi, 3.11 MBq/0.1 mL) was injected intravenously.Other non-pretargeted control animals received ¹⁸F alone (150 μCi, 5.5MBq), Al¹⁸F complex alone (150 μCi, 5.55 MBq), the [Al¹⁸F] IMP 449peptide alone (84 μCi, 3.11 MBq), or [¹⁸F]FDG (150 μXi, 5.55 MBq). ¹⁸Fand [¹⁸F]FDG were obtained on the day of use from IBA Molecular(Somerset, N.J.). Animals receiving [¹⁸F]FDG were fasted overnight, butwater was given ad libitum.

At 1.5 h after the radiotracer injection, animals were anesthetized,bled intracardially, and necropsied. Tissues were weighed and countedtogether with a standard dilution prepared from each of the respectiveproducts. Due to the short physical half-life of ¹⁸F, standards wereinterjected between each group of tissues from each animal. Uptake inthe tissues is expressed as the counts per gram divided by the totalinjected activity to derive the percent-injected dose per gram (% ID/g).

Two types of imaging studies were performed. In one set, 3 nude micebearing small LS174T subcutaneous tumors received either the pretargeted[Al¹⁸F] IMP 449, [Al¹⁸F] IMP 449 alone (not pretargeted), both at 135μCi (5 MBq; 0.1 nmol), or [¹⁸F]FDG (135 μCi, 5 MBq). At 2 h after theintravenous radiotracer injection, the animals were anesthetized with amixture of O₂/N₂O and isoflurane (2%) and kept warm during the scan,performed on an INVEON® animal PET scanner (Siemens PreclinicalSolutions, Knoxyille, Tenn.).

Representative coronal cross-sections (0.8 mm thick) in a plane locatedapproximately in the center of the tumor were displayed, withintensities adjusted until pixel saturation occurred in any region ofthe body (excluding the bladder) and without background adjustment.

In a separate dynamic imaging study, a single LS174T-bearing nude mousethat was given the TF2 bsMAb 16 h earlier was anesthetized with amixture of O₂/N₂O and isoflurane (2%), placed supine on the camera bed,and then injected intravenously with 219 μCi (8.1 MBq) [Al¹⁸F] IMP 449(0.16 nmol). Data acquisition was immediately initiated over a period of120 minutes. The scans were reconstructed using OSEM3D/MAP. Forpresentation, time-frames ending at 5, 15, 30, 60, 90, and 120 min weredisplayed for each cross-section (coronal, sagittal, and transverse).For sections containing tumor, at each interval the image intensity wasadjusted until pixel saturation first occurred in the tumor. Imageintensity was increased as required over time to maintain pixelsaturation within the tumor. Coronal and sagittal cross-sections withouttumor taken at the same interval were adjusted to the same intensity asthe transverse section containing the tumor. Background activity was notadjusted.

Results

While ¹⁸F alone and [Al¹⁸F] complexes had similar uptake in all tissues,considerable differences were found when the complex was chelated to IMP449 (Table 4). The most striking differences were found in the uptake inthe bone, where the non-chelated ¹⁸F was 60- to nearly 100-fold higherin the scapula and ˜200-fold higher in the spine. This distribution isexpected since ¹⁸F, or even a metal-fluoride complex, is known toaccrete in bone (Franke et al. 1972, Radiobiol. Radiother. (Berlin)13:533). Higher uptake was also observed in the tumor and intestines aswell as in muscle and blood. The chelated [Al¹⁸F] IMP 449 hadsignificantly lower uptake in all the tissues except the kidneys,illustrating the ability of the chelate-complex to be removedefficiently from the body by urinary excretion.

Pretargeting the [Al¹⁸F] IMP 449 using the TF2 anti-CEA bsMAb shifteduptake to the tumor, increasing it from 0.20±0.05 to 6.01±1.72% injecteddose per gram at 1.5 h, while uptake in the normal tissues was similarto the [Al¹⁸F] IMP 449 alone. Tumor/nontumor ratios were 146±63, 59±24,38±15, and 2.0±1.0 for the blood, liver, lung, and kidneys,respectively, with other tumor/tissue ratios >100:1 at this time.Although both ¹⁸F alone and [Al¹⁸F] alone had higher uptake in the tumorthan the chelated [Al¹⁸F] IMP 449, yielding tumor/blood ratios of6.7±2.7 and 11.0±4.6 vs. 5.1±1.5, respectively, tumor uptake andtumor/blood ratios were significantly increased with pretargeting (all Pvalues <0.001).

Biodistribution was also compared to the most commonly used tumorimaging agent, [¹⁸F]FDG, which targets tissues with high glucoseconsumption and metabolic activity (Table 4). Its uptake was appreciablyhigher than the [Al¹⁸F] IMP 449 in all normal tissues, except thekidney. Tumor uptake was similar for both the pretargeted [Al¹⁸F] IMP449 and [¹⁸F]FDG, but because of the higher accretion of [¹⁸F]FDG inmost normal tissues, tumor/nontumor ratios with [¹⁸F]FDG weresignificantly lower than those in the pretargeted animals (all P values<0.001).

TABLE 4 Biodistribution of TF2-pretargeted [A1¹⁸F] IMP 449 and othercontrol ¹⁸F-labeled agents in nude mice bearing LS174T human colonicxenografts. For pretargeting, animals were given TF2 16 h before theinjection of the [A1¹⁸F] IMP 449. All injections were administeredintravenously. Percent Injected Dose Per Gram (Mean ± SD) at 1.5 hrPost-Injection [A1¹⁸F] IMP TF2-pretargeted ¹⁸F alone [A1¹⁸F] alone 449alone [A1¹⁸F] IMP 449 [¹⁸F]FDG Tumor 1.02 ± 0.45 1.38 ± 0.39 0.20 ± 0.056.01 ± 1.72 7.25 ± 2.54 Liver 0.11 ± 0.02 0.12 ± 0.02 0.08 ± 0.03 0.11 ±0.03 1.34 ± 0.36 Spleen 0.13 ± 0.06 0.10 ± 0.03 0.08 ± 0.02 0.08 ± 0.022.62 ± 0.73 Kidney 0.29 ± 0.07 0.25 ± 0.07 3.51 ± 0.56 3.44 ± 0.99 1.50± 0.61 Lung 0.26 ± 0.08 0.38 ± 0.19 0.11 ± 0.03 0.17 ± 0.04 3.72 ± 1.48Blood 0.15 ± 0.03 0.13 ± 0.03 0.04 ± 0.01 0.04 ± 0.02 0.66 ± 0.19Stomach 0.21 ± 0.13 0.15 ± 0.05 0.20 ± 0.32 0.12 ± 0.18 2.11 ± 1.04Small Int. 1.53 ± 0.33 1.39 ± 0.34 0.36 ± 0.23 0.27 ± 0.10 1.77 ± 0.61Large Int. 1.21 ± 0.13 1.78 ± 0.70 0.05 ± 0.04 0.03 ± 0.01 2.90 ± 0.79Scapula 6.13 ± 1.33 9.83 ± 2.31 0.08 ± 0.06 0.04 ± 0.02 10.63 ± 5.88 Spine 19.88 ± 2.12  19.03 ± 2.70  0.13 ± 0.14 0.08 ± 0.03 4.21 ± 1.79Muscle 0.16 ± 0.05 0.58 ± 0.36 0.06 ± 0.05 0.10 ± 0.20 4.35 ± 3.01 Brain0.15 ± 0.06 0.13 ± 0.03 0.01 ± 0.01 0.01 ± 0.00 10.71 ± 4.53  Tumor wt(g) 0.29 ± 0.07 0.27 ± 0.10 0.27 ± 0.08 0.33 ± 0.11 0.25 ± 0.21 N 6 7 87 5

Several animals were imaged to further analyze the biodistribution of[Al¹⁸F] IMP 449 alone or [Al¹⁸F] IMP 449 pretargeted with TF2, as well[¹⁸F]FDG. Static images initiated at 2.0 h after the radioactivity wasinjected corroborated the previous tissue distribution data showinguptake almost exclusively in the kidneys (FIG. 1). A 21-mg tumor waseasily visualized in the pretargeted animal, while the animal given the[Al¹⁸F] IMP 449 alone failed to localize the tumor, having only renaluptake. No evidence of bone accretion was observed, suggesting that theAl¹⁸F was bound firmly to IMP 449. This was confirmed in anotherpretargeted animal that underwent a dynamic imaging study that monitoredthe distribution of the [Al¹⁸F] IMP 449 in 5-min intervals over 120minutes (FIG. 2). Coronal and sagittal slices showed primarily cardiac,renal, and some hepatic uptake over the first 5 min, but heart and liveractivity decreased substantially over the next 10 min, while the kidneysremained prominent throughout the study. There was no evidence ofactivity in the intestines or bone over the full 120-min scan. Uptake ina 35-mg LS174T tumor was first observed at 15 min, and by 30 min, thesignal was very clearly delineated from background, with intense tumoractivity being prominent during the entire 120-min scanning.

In comparison, static images from an animal given [¹⁸F]FDG showed theexpected pattern of radioactivity in the bone, heart muscle, and brainobserved previously (McBride et al., 2006, J. Nucl. Med. 47:1678;Sharkey et al., 2008, Radiology 246:497), with considerably morebackground activity in the body (FIG. 1). Tissue uptake measured in the3 animals necropsied at the conclusion of the static imaging studyconfirmed much higher tissue ¹⁸F radioactivity in all tissues (notshown). While tumor uptake with [¹⁸F]FDG was higher in this animal thanin the pretargeted one, tumor/blood ratios were more favorable forpretargeting; and with much less residual activity in the body, tumorvisualization was enhanced by pretargeting.

These studies demonstrate that a hapten-peptide used in pretargetedimaging can be rapidly labeled (60 min total preparation time) with ¹⁸Fby simply forming an aluminum-fluoride complex that can then be bound bya suitable chelate and incorporated into the hapten-peptide. This can bemade more general by simply coupling the [Al¹⁸F]-chelate to any moleculethat can be attached to the chelating moiety and be subsequentlypurified. In preferred embodiments, the percentage incorporation oflabel and specific activity of the labeled compound are sufficient thatpurification of the labeled molecule is not necessary.

This report describes a direct, facile, and rapid method of binding ¹⁸Fto various compounds via an aluminum conjugate. The [Al¹⁸F] peptide wasstable in vitro and in vivo when bound by a NOTA-based chelate. Yieldswere within the range found with conventional ¹⁸F labeling procedures.These results further demonstrate the feasibility of PET imaging usingmetal ¹⁸F chelated to a wide variety of targeting molecules.

Example 11 Preparation and Labeling of IMP 460 with Al—¹⁸F

IMP 460 NODA-Ga-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:26) waschemically synthesized. The NODA-Ga ligand was purchased from CHEMATECH®and attached on the peptide synthesizer like the other amino acids. Thepeptide was synthesized on Sieber amide resin with the amino acids andother agents added in the following order Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Alm removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Alm removal, Fmoc-D-AIa-OH, and NODA-GA(tBu)₃. The peptidewas then cleaved and purified by HPLC to afford the product. HRMSC₆₁H₉₂N₁₈₀₁₈.

Radiolabeling of IMP 460

IMP 460 (0.0020 g) was dissolved in 732 μL, pH 4, 0.1M NaOAc. The ¹⁸Fwas purified as described above, neutralized with glacial acetic acidand mixed with the Al solution. The peptide solution, 20 μL was thenadded and the solution was heated at 99° C. for 25 min. The crudeproduct was then purified on a WATERS® HLB column. The [Al¹⁸F] labeledpeptide was in the 1:1 EtOH/H₂O column eluent. The reverse phase HPLCtrace in 0.1% TFA buffers showed a clean single HPLC peak at theexpected location for the labeled peptide (not shown).

Example 12 Synthesis and Labeling of IMP 461 and IMP 462 NOTA-ConjugatedPeptides

The simplest possible NOTA ligand (protected for peptide synthesis) wasprepared and incorporated into two peptides for pretargeting—IMP 461 andIMP 462.

Synthesis of Di-t-butyl-NOTA

NO₂AtBu (0.501 g 1.4×10⁻³ mol) was dissolved in 5 mL anhydrousacetonitrile. Benzyl-2-bromoacetate (0.222 mL, 1.4×10⁻³ mol) was addedto the solution followed by 0.387 g of anhydrous K₂CO₃. The reaction wasallowed to stir at room temperature overnight. The reaction mixture wasfiltered and concentrated to obtain 0.605 g (86% yield) of the benzylester conjugate. The crude product was then dissolved in 50 mL ofisopropanol, mixed with 0.2 g of 10% Pd/C (under Ar) and placed under 50psi H₂ for 3 days. The product was then filtered and concentrated undervacuum to obtain 0.462 g of the desired product ESMS MH⁻ 415.

Synthesis of IMP 461

The peptide was synthesized on Sieber amide resin with the amino acidsand other agents added in the following order Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Ala-OH, and Bis-t-butylNOTA-OH. Thepeptide was then cleaved and purified by HPLC to afford the product IMP461 ESMS MH⁺1294 (NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; SEQ IDNO:27).

Synthesis of IMP 462

The peptide was synthesized on Sieber amide resin with the amino acidsand other agents added in the following order Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Asp(But)-OH, and Bis-t-butylNOTA-OH.The peptide was then cleaved and purified by HPLC to afford the productIMP 462 ESMS MH⁺1338 (NOTA-D-Asp-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; SEQ IDNO:28).

¹⁸F Labeling of IMP 461 & IMP 462

The peptides were dissolved in pH 4.13, 0.5 M NaOAc to make a 0.05 Mpeptide solution, which was stored in the freezer until needed. The F-18was received in 2 mL of water and trapped on a SEP-PAK® Light, WATERS®ACCELL™ Plus QMA Cartridge. The ¹⁸F was eluted from the column with 200μL aliquots of 0.4 M KHCO₃. The bicarbonate was neutralized to ˜pH 4 bythe addition of 10 μL of glacial acetic acid to the vials before theaddition of the activity. A 100 μL aliquot of the purified ¹⁸F solutionwas removed and mixed with 3 μL, 2 mM Al in pH 4, 0.1M NaOAc. Thepeptide, 10 μL (0.05 M) was added and the solution was heated at ˜100°C. for 15 min. The crude reaction mixture was diluted with 700 μL DIwater and placed on an HLB column and after washing the ¹⁸F was elutedwith 2×100 μL of 1:1 EtOH/H₂O to obtain the purified ¹⁸F-labeledpeptide.

Example 13 Preparation and ¹⁸F Labeling of IMP 467

IMP 467 C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:29)

Tetra tert-butyl C-NETA-succinyl was produced. The tert-Butyl{4-[2-(Bis-(tert-butyoxycarbonyl)methyl-3-(4-nitrophenyl)propyl]-7-tert-butyoxycarbonyl[1,4,7] triazanonan-1-yl} was prepared as described in Chong et al. (J.Med. Chem. 2008, 51:118-125).

The peptide, IMP 467 C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂(SEQ ID NO:29) was made on Sieber Amide resin by adding the followingamino acids to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, the Aloc was cleaved Fmoc-D-Tyr(But)-OH,Aloc-D-Lys(Fmoc)—OH, Trt-HSG-OH, the Aloc was cleaved,tert-Butyl{4-[Bis-(tert-butoxycarbonylmethyl)amino)-3-(4-succinylamidophenyl)propyl]-7-tert-butoxycarbonylmethyl[1,4,7]triazanonan-1-yl}acetate.The peptide was then cleaved from the resin and purified by RP-HPLC toyield 6.3 mg of IMP 467. The crude peptide was purified by highperformance liquid chromatography (HPLC) using a C18 column.

Radiolabeling

A 2 mM solution of IMP 467 was prepared in pH 4, 0.1M NaOAc. The ¹⁸F,139 mCi, was eluted through a WATERS® ACCELL™ Plus SEP-PAK® Light QMAcartridge and the ¹⁸F was eluted with 1 mL of 0.4 M KHCO₃. The labeledIMP 467 was purified by HLB RP-HPLC. The RP-HPLC showed two peakseluting (not shown), which are believed to be diastereomers of Al¹⁸F IMP467. Supporting this hypothesis, there appeared to be someinterconversion between the two HLB peaks when IMP 467 was incubated at37° C. (not shown). In pretargeting techniques as discussed below, sincethe Al¹⁸F-chelator complex is not part of the hapten site for antibodybinding, the presence of diastereomers does not appear to affecttargeting of the ¹⁸F-labeled peptide to diseased tissues.

Comparison of Yield of Radiolabeled Peptides

In an attempt to improve labeling yields while maintaining in vivostability, 3 NOTA derivatives of pretargeting peptide were synthesized(IMP 460, IMP 461 and IMP 467). Of these, IMP 467 nearly doubled thelabeling yields of the other peptides (Table 5). All of the labelingstudies in Table 5 were performed with the same number of moles ofpeptide and aluminum. The results shown in Table 5 represent anexemplary labeling experiment with each peptide.

The ¹⁸F-labeling yield of IMP 467 was ˜70% when only 40 nmol (˜13-foldless than IMP 449) was used with 1.3 GBq (35 mCi) of ¹⁸F, indicatingthis ligand has improved binding properties for the Al¹⁸F complex. Byenhancing the kinetics of ligand binding, yields were substantiallyimproved (average 65-75% yield), while using fewer moles of IMP 467 (40nmol), relative to IMP 449 (520 nmol, 44% yield).

TABLE 5 Comparison of yields of different NOTA containing peptidesPeptide Yield IMP 449 44% IMP 460 5.8% IMP 461 31% IMP 467 87%

Example 14 Pretargeted Biodistribution With ¹⁸F-Labeled IMP 467 inLS174T Tumor Bearing Nude Mice

The ¹⁸F-labeled IMP 467 peptide, prepared in the same manner asdescribed above, was diluted for injection into LS174T tumor bearingnude mice. Al¹⁸F-IMP 467 was prepared and injected in nude mice thatwere necropsied 1.5 h later. Table 6 shows the biodistribution of¹⁸F-labeled IMP 467 using a TF2 pretargeting protocol. The peptide alonecleared quickly from the blood and body, similar to IMP 449 (not shown).The low uptake in the bone compared to ¹⁸F or Al¹⁸F illustrates thestability of the Al¹⁸F chelate and its suitability for pretargeting (notshown). As with IMP 449, using the TF2 bispecific antibody andpretargeting the distribution of labeled IMP 467 was mainly limited totumor and kidney, with very little distribution to bone or other normaltissues (Table 6). Imaging studies with ¹⁸F-labeled IMP 467 are reportedbelow. The data indicate that AlF-18 IMP 467 was stable in-vivo and thepeptide targeted the antibody on the tumor surface.

TABLE 6 TF2 Pretargeted Biodistribution in LS174T Tumor Bearing NudeMice: STD STD STD STD Tissue n Weight WT % ID/g % ID/g % ID/org % ID/orgT/NT T/NT Tumor 6 0.741 0.522 3.415 1.265 2.742 2.112 1.0 0.0 Liver 61.510 0.475 0.175 0.086 0.247 0.087 22.6 12.9 Spleen 6 0.131 0.075 0.3260.261 0.036 0.020 16.3 13.3 Kidney 6 0.160 0.027 3.098 0.647 0.484 0.0761.1 0.3 Lung 6 0.178 0.032 0.204 0.059 0.035 0.011 17.0 5.0 Blood 60.207 0.006 0.153 0.100 0.252 0.145 28.2 13.8 Stomach 6 0.443 0.0890.186 0.148 0.079 0.053 23.3 9.4 Small 6 1.086 0.121 0.338 0.125 0.3590.117 10.5 2.9 Int. Large 6 0.804 0.116 0.115 0.047 0.093 0.045 34.521.1 Int. Scapula 6 0.154 0.040 0.123 0.020 0.019 0.005 28.0 10.9 Spine6 0.199 0.022 0.503 0.372 0.101 0.082 9.4 6.5 Muscle 6 0.088 0.021 0.2000.237 0.014 0.014 56.3 64.3 Brain 6 0.329 0.049 0.013 0.002 0.004 0.000260.4 104.0

Example 15 Factors Affecting Yield and Stability of IMP 467 Labeling

Peptide Concentration

To examine the effect of varying peptide concentration on yield, theamount of binding of Al¹⁸F to peptide was determined in a constantvolume (63 μL) with a constant amount of Al³⁺ (6 nmol) and ¹⁸F, butvarying the amount of peptide added. The yield of labeled peptide IMP467 decreased with a decreasing concentration of peptide as follows: 40nmol peptide (82% yield); 30 nmol (79% yield); 20 nmol (75% yield); 10nmol (49% yield). Thus, varying the amount of peptide between 20 and 40nmol had little effect on yield with IMP 467. However, a decreased yieldwas observed starting at 10 nmol of peptide in the labeling mix.

Aluminum Concentration

When IMP 467 was labeled in the presence of increasing amounts of Al³⁺(0, 5, 10, 15, 20 μL of 2 mM Al in pH 4 acetate buffer and keeping thetotal volume constant), yields of 3.5%, 80%, 77%, 78% and 74%,respectively, were achieved. These results indicated that (a)non-specific binding of ¹⁸F to this peptide in the absence of Al³⁺ isminimal, (b) 10 nmol of Al³⁺ was sufficient to allow for maximum¹⁸F-binding, and (c) higher amounts of Al³⁺did not reduce bindingsubstantially, indicating that there was sufficient chelation capacityat this peptide concentration.

Kinetics of Al¹⁸F IMP 467 Radiolabeling

Kinetic studies showed that binding was complete within 5 min at 107° C.(5 min, 68%; 10 min, 61%; 15 min, 71%; and 30 min, 75%) with onlymoderate increases in isolated yield with reaction times as long as 30min.

A radiolabeling reaction of IMP 467 performed at 50° C. showed that nobinding was achieved at the lower temperature.

Effect of pH

The optimal pH for labeling was between 4.3 and 5.5. Yield ranged from54% at pH 2.88; 70-77% at pH 3.99; 70% at pH 5; 41% at pH 6 to 3% at pH7.3. The process could be expedited by eluting the ¹⁸F⁻ from the anionexchange column with nitrate or chloride ion instead of carbonate ion,which eliminates the need for adjusting the eluent to pH 4 with glacialacetic acid before mixing with the AlCl₃.

High-Dose Radiolabeling of IMP 467

Five microliters of 2 mM Al³⁺ stock solution were mixed with 50 μL of¹⁸F 1.3 GBq (35 mCi) followed by the addition of 20 μL of 2 mM IMP 467in 0.1 mM, pH 4.1 acetate buffer. The reaction solution was heated to104° C. for 15 min and then purified on an HLB column (−10 min) asdescribed above, isolating 0.68 GBq (18.4 mCi) of the purified peptidein 69% radiochemical yield with a specific activity of 17 GBq/μmol (460Ci/mmol). The reaction time was 15 mM and the purification time was 12min. The reaction was started 10 mM after the 1.3 GBq (35 mCi) ¹⁸F waspurified, so the total time from the isolation of the ¹⁸F to thepurified final product was 37 min with a 52% yield without correctingfor decay.

Human Serum Stability Test

An aliquot of the HLB purified peptide (˜30 μL) was diluted with 200 μLhuman serum (previously frozen) and placed in the 37° C. HPLC samplechamber. Aliquots were removed at various time points and analyzed byHPLC. The HPLC analysis showed very high stability of the ¹⁸F-labeledpeptides in serum at 37° C. for at least five hours (not shown). Therewas no detectable breakdown of the ¹⁸F-labeled peptide after a five hourincubation in serum (not shown).

The IMP 461 and IMP 462 ligands have two carboxyl groups available tobind the aluminum whereas the NOTA ligand in IMP 467 had four carboxylgroups. The serum stability study showed that the complexes with IMP 467were stable in serum under conditions replicating in vivo use. Further,the in vivo biodistribution studies with labeled IMP 467 show that the¹⁸F—Al labeled peptide is stable under actual in vivo conditions.

Peptides can be labeled with ¹⁸F rapidly (30 min) and in high yield byforming Al¹⁸F complexes that can be bound to a NOTA ligand on a peptideand at a specific activity of at least 17 GBq/μmol, without requiringHPLC purification. The Al¹⁸F NOTA-peptides are stable in serum and invivo. Modifications of the NOTA ligand can lead to improvements in yieldand specific activity, while still maintaining the desired in vivostability of the Al¹⁸F-NOTA complex, and being attached to a hydrophiliclinker aids in the renal clearance of the peptide. Further, this methodavoids the dry-down step commonly used to label peptides with ¹⁸F. Asshown in the following Examples, this new ¹⁸F-labeling method isapplicable to labeling of a broad spectrum of targeting peptides.

Optimized Labeling of Al¹⁸F IMP 467

Optimized conditions for ¹⁸F labeling of IMP 467 were identified. Theseconsisted of eluting ¹⁸F-fluoride with commercial sterile saline (pH5-7), mixing with 20 nmol of AlCl₃ and 40 nmol IMP 467 in pH 4 acetatebuffer in a total volume of 100 μL, heating to 102° C. for 15 min, andperforming SPE separation. High-yield (85%) and high specific activity(115 GBq/μ-mol) were obtained with IMP 467 in a single step, 30-minprocedure after a simple solid-phase extraction (SPE) separation withoutthe need for HPLC purification. ¹⁸F-IMP 467 was stable in PBS or humanserum, with 2% loss of ¹⁸F⁻ after incubation in either medium for 6 h at37° C.

Concentration and Purification of ¹⁸F⁻

Radiochemical-grade ¹⁸F⁻ needs to be purified and concentrated beforeuse. We examined 4 different SPE purification procedures to process the¹⁸F⁻ prior to its use. Most of the radiolabeling procedures wereperformed using ¹⁸F⁻ prepared by a conventional process. The ¹⁸F in 2 mLof water was loaded onto a SEP-PAK® Light, Waters Accell™ QMA PlusCartridge that was pre-washed with 10 mL of 0.4M KHCO₃, followed by 10mL water. After loading the ¹⁸F onto the cartridge, it was washed with 5mL water to remove any dissolved metal and radiometal impurities. Theisotope was then eluted with ˜1 mL of 0.4M KHCO₃ in several fractions toisolate the fraction with the highest concentration of activity. Theeluted fractions were neutralized with 5 μL of glacial acetic acid per100 μL of solution to adjust the eluent to pH 4-5.

In the second process, the QMA cartridge was washed with 10 mL pH 8.4,0.5 M NaOAc followed by 10 mL DI H₂O. ¹⁸F⁻ was loaded onto the column asdescribed above and eluted with 1 mL, pH 6, 0.05 M KNO₃ in 200-μLfractions with 60-70% of the activity in one of the fractions. No pHadjustment of this solution was needed.

In the third process, the QMA cartridge was washed with 10 mL pH 8.4,0.5 M NaOAc followed by 10 mL DI H₂O. The ¹⁸F⁻ was loaded onto thecolumn as described above and eluted with 1 mL, pH 5-7, 0.154 Mcommercial normal saline in 200-μL fractions with 80% of the activity inone of the fractions. No pH adjustment of this solution was needed.

Finally, we devised a method to prepare a more concentrated andhigh-activity ¹⁸F⁻ solution, using tandem ion exchange. Briefly, Tygontubing (1.27 cm long, 0.64 cm OD) was inserted into a TRICORN™ 5/20column and filled with ˜200 μL of AG 1-X8 resin, 100-200 mesh. The resinwas washed with 6 mL 0.4 M K₂CO₃ followed by 6 mL H₂O. A SEP-PAK® lightWaters ACCELL™ Plus CM cartridge was washed with DI H₂O. Using a syringepump, the crude ¹⁸F⁻ that was received in 5-mL syringe in 2 mL DI H₂Oflowed slowly through the CM cartridge and the TRICORN™ column over ˜5min followed by a 6 mL wash with DI H₂O through both ion-bindingcolumns. Finally, 0.4 M K₂CO₃ was pushed through only the TRICORN™column in 50-μL fractions. Typically, 40 to 60% of the eluted activitywas in one 50-μL fraction. The fractions were collected in 2.0 mLfree-standing screw-cap microcentrifuge tubes containing 5 μL glacialacetic acid to neutralize the carbonate solution. The elution vial withthe most activity was then used as the reaction vial.

Example 16 Synthesis and Labeling of IMP 470

IMP 470 L-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:30)

The peptide, IMP 470 was made on Sieber Amide resin by adding thefollowing amino acids to the resin in the order shown:Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved,Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH. The free amineobtained after the removal of Aloc was reacted with succinic anhydride,to generate a carboxylic acid group at the N-terminus, which isactivated using DIC in DMF and subsequently coupled with tert-butylprotected L-NETA. The peptide was then cleaved from the resin andpurified by RP-HPLC to yield 16.4 mg of IMP 470. A product withmolecular mass 1037.15 corresponding to the peptide without L-NETA andwith retention time 9.001 min was also obtained.

For ¹⁸F labeling, to 3 μL 2 mM AlCl₃ solution was added 40 μL F-18solution [1.736 mCi of ¹⁸F] followed by 20 μL (40 nmol) 2 mM IMP 470solution and heated to 101° C. for 15 minutes. Reverse Phase HPLCanalysis showed two radiolabeled peptide peaks at 26.10% (RT 8.90 min)and 47.29% (RT 9.30 min) and 26.61% of the activity eluted at the voidvolume of the column (2.70 min) (not shown).

The crude labeled peptide was purified by HLB column chromatography,eluting with 1:1 EtOH/H₂O to collect labeled peptide. The 790 μCicollected represents a recovery of 65.83% of labeled peptide.

Sixty μL of the HLB column purified ¹⁸F-labeled peptide were mixed with100 μL human serum and the sample was maintained at 37° C. during theentire RP-HPLC analysis. The ¹⁸F-labeled IMP 470 was stable in serum forat least 4 hours (not shown).

Example 17 Labeling by Addition of ¹⁸F⁻ to a Peptide Pre-Incubated WithAluminum

An HSG containing peptide (IMP 465,NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)—NH₂) (SEQ ID NO:31) linked tomacrocyclic NOTA complexed with aluminum, was successfully labeled withF-18. ¹⁸F incorporation using 40 nmol of IMP 465 was 13.20%. Anintermediate peptide, IMP 461, was made as described above. Then 25.7 mgof IMP 461 was dissolved in 2 mL DI water to which was added 10.2 mgAlCl₃.6H₂O and the resultant solution heated to 100° C. for 1 h. Thecrude reaction mixture was purified by RP-HPLC to yield 19.6 mg of IMP465.

For ¹⁸F labeling, 50 μL ¹⁸F solution [0.702 mCi of ¹⁸F] and 20 μL (40nmol) 2 mM IMP 465 solution (0.1M NaOAc, pH 4.18) was heated to 101° C.for 17 minutes. Reverse Phase HPLC analysis showed 15.38% (RT about 8.60min) of the activity was attached to the peptide and 84.62% of theactivity eluted at the void volume of the column (2.60 min).

In a separate experiment, the percent yield of ¹⁸F-labeled peptide couldbe improved by varying the amount of peptide added. The percent yieldobserved for IMP 465 was 0.27% at 10 nmol peptide, 1.8% at 20 nmol ofpeptide and 49% at 40 nmol of peptide.

IMP 467 showed higher yield than IMP 461 when peptide was pre-incubatedwith aluminum before exposure to ¹⁸F. IMP 467 was incubated withaluminum at room temperature and then frozen and lyophilized. The amountof aluminum added for the pre-incubation was varied.

TABLE 7 Labeling of IMP 467 Pre-Incubated with Aluminum Before ¹⁸F isAdded IMP 467 + Al Premixed, Isolated Frozen and Lyophilized LabelingYield 40 nmol IMP 467 + nmol Al Premix 82% 40 nmol IMP 467 + nmol AlPremix 64% 40 nmol IMP 467 + nmol Al Premix 74% 40 nmol IMP 467 + 6 nmolAl Normal 77% Labeling (Mix Al + ¹⁸F first)

The yields were comparable to those obtained when IMP 467 is labeled byaddition of an Al¹⁸F complex. Thus, ¹⁸F labeling by addition of ¹⁸F to apeptide with aluminum already bound to the chelating moiety is afeasible alternative approach to pre-incubating the metal with ¹⁸F priorto addition to the chelating moiety.

Example 18 Synthesis and Labeling of IMP 468 Bombesin Peptide

The ¹⁸F labeled targeting moieties are not limited to antibodies orantibody fragments, but rather can include any molecule that bindsspecifically or selectively to a cellular target that is associated withor diagnostic of a disease state or other condition that may be imagedby ¹⁸F PET. Bombesin is a 14 amino acid peptide that is homologous toneuromedin B and gastrin releasing peptide, as well as a tumor markerfor cancers such as lung and gastric cancer and neuroblastoma. IMP 468(NOTA-NH—(CH₂)₇CO-Gln-Trp-Val-Trp-Ala-Val-Gly-His-Leu-Met-NH₂; SEQ IDNO:32) was synthesized as a bombesin analogue and labeled with ¹⁸F totarget the gastrin-releasing peptide receptor.

The peptide was synthesized by Fmoc based solid phase peptide synthesison Sieber amide resin, using a variation of a synthetic scheme reportedin the literature (Prasanphanich et al., 2007, PNAS USA 104:12463-467).The synthesis was different in that a bis-t-butyl NOTA ligand was add tothe peptide during peptide synthesis on the resin.

IMP 468 (0.0139 g, 1.02×10⁻⁵ mol) was dissolved in 203 μL of 0.5 M pH4.13 NaOAc buffer. The peptide dissolved but formed a gel on standing sothe peptide gel was diluted with 609 μL of 0.5 M pH 4.13 NaOAc bufferand 406 jiL of ethanol to produce an 8.35×10⁻³ M solution of thepeptide. The ¹⁸F was purified on a QMA cartridge and eluted with 0.4 MKHCO₃ in 200 μL fractions, neutralized with 10 μL of glacial aceticacid. The purified ¹⁸F, 40 μL 1.13 mCi was mixed with 3 μL of 2 mM AlCl₃in pH 4, 0.1M NaOAc buffer. IMP 468 (59.2 μL, 4.94×10⁻⁷ mol) was addedto the Al¹⁸F solution and placed in a 108° C. heating block for 15 min.The crude product was purified on an HLB column, eluted with 2×200 μL of1:1 EtOH/H₂O to obtain the purified ¹⁸F-labeled peptide in 34% yield.

Example 19 Imaging of Tumors Using ¹⁸F Labeled Bombesin

A NOTA-conjugated bombesin derivative (IMP 468) was prepared asdescribed above. We began testing its ability to block radiolabeledbombesin from binding to PC-3 cells as was done by Prasanphanich et al.(PNAS 104:12462-12467, 2007). Our initial experiment was to determine ifIMP 468 could specifically block bombesin from binding to PC-3 cells. Weused IMP 333 as a non-specific control. In this experiment, 3×10⁶ PC-3cells were exposed to a constant amount (˜50,000 cpms) of ¹²⁵I-Bombesin(Perkin-Elmer) to which increasing amounts of either IMP 468 or IMP 333was added. A range of 56 to 0.44 nM was used as our inhibitoryconcentrations.

The results showed that we could block the binding of ¹²⁵I-BBN with IMP468 but not with the control peptide (IMP 333) (not shown), thusdemonstrating the specificity of IMP 468. Prasanphanich indicated anIC₅₀ for their peptide at 3.2 nM, which is approximately 7-fold lowerthan what we found with IMP 468 (21.5 nM).

This experiment was repeated using a commercially available BBN peptide.We increased the amount of inhibitory peptide from 250 to 2 nM to blockthe ¹²⁵I-BBN from binding to PC-3 cells. We observed very similarIC₅₀-values for IMP 468 and the BBN positive control with an IC₅₀-valuehigher (35.9 nM) than what was reported previously (3.2 nM) but close towhat the BBN control achieved (24.4 nM).

To examine in vivo targeting, the distribution of Al¹⁸F IMP 468 wasexamined in scPC3 prostate cancer xenograft bearing nude male mice;alone vs. blocked with bombesin. For radiolabeling, aluminum chloride(10 μL, 2 min), 51.9 mCi of ¹⁸F (from QMA cartridge), acetic acid, and60 μL of IMP 468 (8.45 mM in ethanol/NaOAc) were heated at 100° C. for15 min. The reaction mixture was purified on reverse phase HPLC.Fractions 40 and 41 (3.56, 1.91 mCi) were pooled and applied to HLBcolumn for solvent exchange. The product was eluted in 800 μL (3.98 mCi)and 910 μCi remained on the column. ITLC developed in saturated NaClshowed 0.1% unbound activity.

A group of six tumor-bearing mice were injected with [Al¹⁸F] IMP 468(167 μCi, ˜9×10⁻¹° mol) and necropsied 1.5 h later. Another group of sixmice were injected iv with 100 μg (6.2×10⁻⁸ mol) of bombesin 18 minbefore administering [Al¹⁸F] IMP 468. The second group was alsonecropsied 1.5 h post injection. The data shows specific targeting ofthe tumor with [Al¹⁸F] IMP 468 (FIG. 3). Tumor uptake of the peptide isreduced when bombesin was given 18 min before the [Al¹⁸F] IMP 468 (FIG.3). Biodistribution data indicates in vivo stability of [Al¹⁸F] IMP 468for at least 1.5 h (not shown).

Larger tumors showed higher uptake of [Al¹⁸F] IMP 468, possibly due tohigher receptor expression in larger tumors (not shown). Thebiodistribution data showed [Al¹⁸F] IMP 468 tumor targeting that was inthe same range as reported for the same peptide labeled with ⁶⁸Ga byPrasanphanich et al. (not shown). The results demonstrate that the ¹⁸Fpeptide labeling method can be used in vivo to target receptors that areupregulated in tumors, using targeting molecules besides antibodies. Inthis case, the IMP 468 targeting took advantage of a naturally occurringligand-receptor interaction. The tumor targeting was significant with aP value of P=0.0013. Many such ligand-receptor pairs are known and anysuch targeting interaction may form the basis for ¹⁸F-imaging, using themethods described herein.

Example 20 Synthesis and Labeling of Somatostatin Analog IMP 466

Somatostatin is another non-antibody targeting peptide that is of usefor imaging the distribution of somatostatin receptor protein.¹²³I-labeled octreotide, a somatostatin analog, has been used forimaging of somatostatin receptor expressing tumors (e.g., Kvols et al.,1993, Radiology 187:129-33; Leitha et al., 1993, J Nucl Med34:1397-1402). However, ¹²³I has not been of extensive use for imagingbecause of its expense, short physical half-life and the difficulty ofpreparing the radiolabeled compounds. The ¹⁸F-labeling methods describedherein are preferred for imaging of somatostatin receptor expressingtumors.

(SEQ ID NO: 33) IMP 466 NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl

A NOTA-conjugated derivative of the somatostatin analog octreotide (IMP466) was made by standard Fmoc based solid phase peptide synthesis toproduce a linear peptide. The C-terminal Throl residue is threoninol.The peptide was cyclized by treatment with DMSO overnight. The peptide,0.0073 g, 5.59×10⁻⁶ mol was dissolved in 111.9 μL of 0.5 M pH 4 NaOAcbuffer to make a 0.05 M solution of IMP 466. The solution formed a gelover time so it was diluted to 0.0125 M by the addition of more 0.5 MNaOAc buffer.

¹⁸F was purified and concentrated with a QMA cartridge to provide 200 μLof ¹⁸F in 0.4 M KHCO₃. The bicarbonate solution was neutralized with 10μL of glacial acetic acid. A 40 aliquot of the neutralized ¹⁸F eluentwas mixed with 3 μL of 2 mM AlCl₃, followed by the addition of 40 μL of0.0125 M IMP 466 solution. The mixture was heated at 105° C. for 17 min.The reaction was then purified on a Waters 1 cc (30 mg) HLB column byloading the reaction solution onto the column and washing the unbound¹⁸F away with water (3 mL) and then eluting the radiolabeled peptidewith 2×200 μL 1:1 EtOH water. The yield of the radiolabeled peptideafter HLB purification was 34.6%.

Effect of Ionic Strength

To lower the ionic strength of the reaction mixture escalating amountsof acetonitrile were added to the labeling mixture (final concentration:0-80%). The yield of radiolabeled IMP 466 increased with increasingconcentration of acetonitrile in the medium. The optimal radiolabelingyield (98%) was obtained in a final concentration of 80% acetonitrile,despite the increased volume (500 μL in 80% vs. 200 μL in 0%acetonitrile). In 0% acetonitrile the radiolabeling yield ranged from36% to 55% in three experiments.

Example 21 Imaging of Neuroendocrine Tumors with an ¹⁸F- and⁶⁸Ga-Labeled IMP 466

Studies were performed to compare the PET images obtained using an ¹⁸Fversus ⁶⁸Ga-labeled somatostatin analogue peptide and direct targetingto somatostatin receptor expressing tumors.

Methods

¹⁸F Labeling

IMP 466 was synthesized and ¹⁸F-labeled by a variation of the methoddescribed in the Example above. A QMA SEPPAK® light cartridge (Waters,Milford, Mass.) with 2-6 GBq ¹⁸F (BV Cyclotron VU, Amsterdam, TheNetherlands) was washed with 3 mL metal-free water. ¹⁸F was eluted fromthe cartridge with 0.4 M KHCO₃ and fractions of 200 μL were collected.The pH of the fractions was adjusted to pH 4, with 10 μL metal-freeglacial acid. Three μL of 2 mM AlCl₃ in 0.1M sodium acetate buffer, pH 4were added. Then, 10-50 μL IMP 466 (10 mg/mL) were added in 0.5 M sodiumacetate, pH 4.1. The reaction mixture was incubated at 100° C. for 15min unless stated otherwise. The radiolabeled peptide was purified onRP-HPLC. The ¹⁸F-IMP 466-containing fractions were collected and dilutedtwo-fold with H₂O and purified on a 1-cc Oasis HLB cartridge (Waters,Milford, Mass.) to remove acetonitrile and TFA. In brief, the fractionwas applied on the cartridge and the cartridge was washed with 3 mL H₂O.The radiolabeled peptide was then eluted with 2×200 μL 50% ethanol. Forinjection in mice, the peptide was diluted with 0.9% NaCl. A maximumspecific activity of 45,000 GBq/mmol was obtained.

⁶⁸Ga Labeling

IMP 466 was labeled with ⁶⁸GaCl₃ eluted from a TiO₂-based 1,110 MBq⁶⁸Ge/⁶⁸Ga generator (Cyclotron Co. Ltd., Obninsk, Russia) using 0.1Multrapure HCl (J. T. Baker, Deventer, The Netherlands). IMP 466 wasdissolved in 1.0 M HEPES buffer, pH 7.0. Four volumes of ⁶⁸Ga eluate(120-240 MBq) were added and the mixture was heated at 95° C. for 20min. Then 50 mM EDTA was added to a final concentration of 5 mM tocomplex the non-incorporated ⁶⁸Ga³⁺. The ⁶⁸Ga-labeled IMP 466 waspurified on an Oasis HLB cartridge and eluted with 50% ethanol.

Octanol-Water Partition Coefficient (Log P_(oct/water))

To determine the lipophilicity of the radiolabeled peptides,approximately 50,000 dpm of the radiolabeled peptide was diluted in 0.5mL phosphate-buffered saline (PBS). An equal volume of octanol was addedto obtain a binary phase system. After vortexing the system for 2 min,the two layers were separated by centrifugation (100×g, 5 min). Three100 μL samples were taken from each layer and radioactivity was measuredin a well-type gamma counter (Wallac Wizard 3″, Perkin-Elmer, Waltham,Mass.).

Stability

Ten μL of the ¹⁸F-labeled IMP 466 was incubated in 500 μL of freshlycollected human serum and incubated for 4 h at 37° C. Acetonitrile wasadded and the mixture was vortexed followed by centrifugation at 1000×gfor 5 min to precipitate serum proteins. The supernatant was analyzed onRP-HPLC as described above.

Cell Culture

The AR42J rat pancreatic tumor cell line was cultured in Dulbecco'sModified Eagle's Medium (DMEM) medium (Gibco Life Technologies,Gaithersburg, Md., USA) supplemented with 4500 mg/L D-glucose, 10% (v/v)fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin and 100 μg/mLstreptomycin. Cells were cultured at 37° C. in a humidified atmospherewith 5% CO₂.

IC₅₀ Determination

The apparent 50% inhibitory concentration (IC₅₀) for binding thesomatostatin receptors on AR42J cells was determined in a competitivebinding assay using ¹⁹F-IMP 466, ⁶⁹Ga-IMP 466 or ¹¹⁵In-DTPA-octreotideto compete for the binding of ¹¹¹In-DTPA-octreotide.

¹⁹F-IMP 466 was formed by mixing an aluminium fluoride (AlF) solution(0.02 M AlCl₃ in 0.5 M NaAc, pH 4, with 0.1M NaF in 0.5 M NaAc, pH 4)with IMP 466 and heating at 100° C. for 15 min. The reaction mixture waspurified by RP-HPLC on a C-18 column as described above.

⁶⁹Ga-IMP 466 was prepared by dissolving gallium nitrate (2.3×10⁻⁸ mol)in 30 μL mixed with 20 μL IMP 466 (1 mg/mL) in 10 mM NaAc, pH 5.5, andheated at 90° C. for 15 min. Samples of the mixture were used withoutfurther purification.

¹¹⁵In-DTPA-octreotide was made by mixing indium chloride (1×10⁻⁵ mol)with 10 μL DTPA-octreotide (1 mg/mL) in 50 mM NaAc, pH 5.5, andincubated at room temperature (RT) for 15 min. This sample was usedwithout further purification. ¹¹¹¹n-DTPA-octreotide (OCTREOSCAN®) wasradiolabeled according to the manufacturer's protocol.

AR42J cells were grown to confluency in 12-well plates and washed twicewith binding buffer (DMEM with 0.5% bovine serum albumin). After 10 minincubation at RT in binding buffer, ¹⁹F-IMP 466, ⁶⁹Ga-IMP 466 or¹¹⁵In-DTPA-octreotide was added at a final concentration ranging from0.1-1000 nM, together with a trace amount (10,000 cpm) of¹¹¹In-DTPA-octreotide (radiochemical purity >95%). After incubation atRT for 3 h, the cells were washed twice with ice-cold PBS. Cells werescraped and cell-associated radioactivity was determined. Under theseconditions, a limited extent of internalization may occur. We thereforedescribe the results of this competitive binding assay as “apparentIC₅₀” values rather than IC₅₀. The apparent IC₅₀ was defined as thepeptide concentration at which 50% of binding without competitor wasreached.

Biodistribution Studies

Male nude BALB/c mice (6-8 weeks) were injected subcutaneously in theright flank with 0.2 mL AR42J cell suspension of 10⁷ cells/mL.Approximately two weeks after tumor cell inoculation when tumors were5-8 mm in diameter, 370 kBq ¹⁸F or ⁶⁸Ga-labeled IMP 466 was administeredintravenously (n=5). Separate groups (n=5) were injected with a1,000-fold molar excess of unlabeled IMP 466. One group of three micewas injected with unchelated [Al¹⁸F]. All mice were killed by CO₂/O₂asphyxiation 2 h post-injection (p.i.). Organs of interest werecollected, weighed and counted in a gamma counter. The percentage of theinjected dose per gram tissue (% ID/g) was calculated for each tissue.The animal experiments were approved by the local animal welfarecommittee and performed according to national regulations.

PET/CT Imaging

Mice with s.c. AR42J tumors were injected intravenously with 10 MBqAl¹⁸F-IMP 466 or ⁶⁸Ga-IMP 466. One and two hours after the injection ofpeptide, mice were scanned on an Inveon animal PET/CT scanner (SiemensPreclinical Solutions, Knoxyille, Tenn.) with an intrinsic spatialresolution of 1.5 mm (Visser et al, JNM, 2009). The animals were placedin a supine position in the scanner. PET emission scans were acquiredover 15 minutes, followed by a CT scan for anatomical reference (spatialresolution 113 μm, 80 kV, 500 μA). Scans were reconstructed using InveonAcquisition Workplace software version 1.2 (Siemens PreclinicalSolutions, Knoxyille, Tenn.) using an ordered set expectationmaximization-3D/maximum a posteriori (OSEM3DIMAP) algorithm with thefollowing parameters: matrix 256×256×159, pixel size 0.43×0.43×0.8 mm³and MAP prior of 0.5 mm.

Results

Effect of Buffer

The effect of the buffer on the labeling efficiency of IMP 466 wasinvestigated. IMP 466 was dissolved in sodium citrate buffer, sodiumacetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at 10mg/mL (7.7 min). The molarity of all buffers was 1M and the pH was 4.1.To 200 μg (153 nmol) of IMP 466 was added 100 μL Al—F-18 (pH 4) andincubated at 100° C. for 15 mM. Radiolabeling yield and specificactivity was determined with RP-HPLC. When using sodium acetate, MES orHEPES, radiolabeling yield was 49%, 44% and 46%, respectively. In thepresence of sodium citrate, no labeling was observed (<1%). When thelabeling reaction was carried out in sodium acetate buffer, the specificactivity of the preparations was 10,000 GBq/mmol, whereas in MES andHEPES buffer a specific activity of 20,500 and 16,500 GBq/mmol wasobtained, respectively.

Effect of AlCl₃ Concentration

Three stock solutions of AlCl₃ in sodium acetate, pH 4.1 were prepared:0.2, 2.0 and 20 mM. From these solutions, 3 μL was added to 200 μL of¹⁸F to form [Al¹⁸F]. To these samples, 153 nmol of peptide was added andincubated for 15 min at 100° C. Radiolabeling yield was 49% afterincubation at a final concentration of 6 nmol AlCl₃. Incubation with 0.6nmol AlCl₃ and 60 nmol AlCl₃ resulted in a strong reduction of theradiolabeling yield: 10% and 6%, respectively.

Effect of Amount of Peptide

The effect of the amount of peptide on the labeling efficiency wasinvestigated. IMP 466 was dissolved in sodium acetate buffer, pH 4.1 ata concentration of 7.7 mM (10 mg/mL) and 38, 153 or 363 nmol of IMP 466was added to 200 [Al¹⁸F] (581-603 MBq). The radiolabeling yieldincreased with increasing amounts of peptide. At 38 nmol, radiolabelingyield ranged from 4-8%, at 153 nmol, the yield had increased to 42-49%and at the highest concentration the radiolabeling yield was 48-52%.

Octanol-Water Partition Coefficient

To determine the lipophilicity of the ¹⁸F and ⁶⁸Ga-labeled IMP 466, theoctanol-water partition coefficients were determined. The logP_(octanol/water) value for the Al¹⁸F-IMP 466 was −2.44±0.12 and that of⁶⁸Ga-IMP 466 was −3.79±0.07, indicating that the ¹⁸F-labeled IMP 466 wasslightly less hydrophilic.

Stability

The ¹⁸F-labeled IMP 466 did not show release of ¹⁸F after incubation inhuman serum at 37° C. for 4 h, indicating excellent stability of theAl¹⁸F-NOTA complex.

IC₅₀ Determination

The apparent IC₅₀ of Al¹⁹F-labeled IMP 466 was 3.6±0.6 nM, whereas thatfor ⁶⁹Ga-labeled IMP 466 was 13±3 nM. The apparent IC₅₀ of the referencepeptide, ¹¹⁵¹n-DTPA-octeotride (OCTREOSCAN®), was 6.3±0.9 nM.

Biodistribution Studies

The biodistribution of both Al¹⁸F-IMP 466 and ⁶⁸Ga-IMP 466 was studiedin nude BALB/c mice with s.c. AR42J tumors at 2 h p.i. (FIG. 4). Al¹⁸Fwas included as a control. Tumor targeting of the ¹⁸F-IMP 466 was highwith 28.3±5.7% ID/g accumulated at 2 h p.i. Uptake in the presence of anexcess of unlabeled IMP 466 was 8.6±0.7% ID/g, indicating that tumoruptake was receptor-mediated. Blood levels were very low (0.10±0.07%ID/g, 2 h pi), resulting in a tumor-to-blood ratio of 299±88. Uptake inthe organs was low, with specific uptake in receptor expressing organssuch as adrenal glands, pancreas and stomach. Bone uptake of Al¹⁸F-IMP466 was negligible as compared to uptake of non-chelated Al¹⁸F(0.33±0.07 vs. 36.9±5.0% ID/g at 2 h p.i., respectively), indicatinggood in vivo stability of the ¹⁸F-labeled NOTA-peptide.

The biodistribution of Al¹⁸F-IMP 466 was compared to that of ⁶⁸Ga-IMP466 (FIG. 4). Tumor uptake of ⁶⁸Ga-IMP 466 (29.2±0.5% ID/g, 2 h pi) wassimilar to that of Al¹⁸F-IMP 466 (p<0.001). Lung uptake of ⁶⁸Ga-IMP 466was two-fold higher than that of ¹⁸F-IMP 466 (4.0±0.9% ID/g vs. 1.9±0.4%ID/g, respectively). In addition, kidney retention of ⁶⁸Ga-IMP 466 wasslightly higher than that of Al¹⁸F-IMP 466 (16.2±2.86% ID/g vs.9.96±1.27% ID/g, respectively.

Fused PET and CT scans are shown in FIG. 5. PET scans corroborated thebiodistribution data. Both Al¹⁸F-IMP 466 and ⁶⁸Ga-IMP 466 showed highuptake in the tumor and retention in the kidneys. The activity in thekidneys was mainly localized in the renal cortex. Again, the Al¹⁸Fproved to be stably chelated by the NOTA chelator, since no bone uptakewas observed.

FIG. 5 clearly shows that the distribution of an ¹⁸F-labeled analog ofsomatostatin (octreotide) mimics that of a ⁶⁸Ga-labeled somatostatinanalog. These results are significant, since ⁶⁸Ga-labeled octreotide PETimaging in human subjects with neuroendocrine tumors has been shown tohave a significantly higher detection rate compared with conventionalsomatostatin receptor scintigraphy and diagnostic CT, with a sensitivityof 97%, a specificity of 92% and an accuracy of 96% (e.g., Gabriel etal., 2007, J Nucl Med 48:508-18). PET imaging with ⁶⁸Ga-labeledoctreotide is reported to be superior to SPECT analysis with¹¹¹In-labeled octreotide and to be highly sensitive for detection ofeven small meningiomas (Henze et al., 2001, J Nucl Med 42:1053-56).Because of the higher energy of ⁶⁸Ga compared with ¹⁸F, it is expectedthat ¹⁸F based PET imaging would show even better spatial resolutionthan ⁶⁸Ga based PET imaging. This is illustrated in FIG. 5 by comparingthe kidney images obtained with ¹⁸F-labeled IMP 466 (FIG. 5, left) vs.⁶⁸Ga-labeled IMP 466 (FIG. 5, right). The PET images obtained with ⁶⁸Gashow more diffuse margins and lower resolution than the images obtainedwith ¹⁸F. These results demonstrate the superior images obtained with¹⁸F-labeled targeting moieties prepared using the methods andcompositions described herein and confirm the utility of the described¹⁸F labeling techniques for non-antibody targeting peptides.

Example 22 Comparison of ⁶⁸Ga and ¹⁸F PET Imaging Using Pretargeting

We compared PET images obtained using ⁶⁸Ga- or ¹⁸F-labeled peptides thatwere pretargeted with the bispecific TF2 antibody, prepared as describedabove. The half-lives of ⁶⁸Ga (t_(1/2)=68 minutes) and ¹⁸F(t_(1/2)=110minutes) match with the pharmacokinetics of the radiolabeled peptide,since its maximum accretion in the tumor is reached within hours.Moreover, ⁶⁸Ga is readily available from ⁶⁸Ge/⁶⁸Ga generators, whereas¹⁸F is the most commonly used and widely available radionuclide in PET.

Methods

Mice with s.c. CEA-expressing LS174T tumors received TF2 (6.0 nmol; 0.94mg) and 5 MBq ⁶⁸Ga-labeled IMP 288 (0.25 nmol) or ¹⁸F-labeled IMP 449(0.25 nmol) intravenously, with an interval of 16 hours between theinjection of the bispecific antibody and the radiolabeled peptide. Oneor two hours after the injection of the radiolabeled peptide, PET/CTimages were acquired and the biodistribution of the radiolabeled peptidewas determined. Uptake in the LS174T tumor was compared with that in ans.c. CEA-negative SK-RC 52 tumor. Pretargeted immunoPET imaging wascompared with ¹⁸F-FDG-PET imaging in mice with an s.c. LS174T tumor andcontralaterally an inflamed thigh muscle.

(SEQ ID NO: 34) IMP 288 DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂

Pretargeting

The bispecific TF2 antibody was made by the DNL method, as describedabove. TF2 is a trivalent bispecific antibody comprising an HSG-bindingFab fragment from the h679 antibody and two CEA-binding Fab fragmentsfrom the hMN-14 antibody. The DOTA-conjugated, HSG-containing peptideIMP 288 was synthesized by peptide synthesis as described above. The IMP449 peptide, synthesized as described above, contains a1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelating moiety tofacilitate labeling with ¹⁸F. As a tracer for the antibody component,TF2 was labeled with ¹²⁵I (Perkin Elmer, Waltham, Mass.) by the iodogenmethod (Fraker and Speck, 1978, Biochem Biophys Res Comm 80:849-57), toa specific activity of 58 MBq/nmol.

Labeling of IMP 288

IMP 288 was labeled with ¹¹¹In (Covidien, Petten, The Netherlands) forbiodistribution studies at a specific activity of 32 MBq/nmol understrict metal-free conditions. IMP 288 was labeled with ⁶⁸Ga eluted froma TiO-based 1,110 MBq ⁶⁸Ge/⁶⁸Ga generator (Cyclotron Co. Ltd., ObninskRussia) using 0.1M ultrapure HCl. Five 1 ml fractions were collected andthe second fraction was used for labeling the peptide. One volume of 1.0M HEPES buffer, pH 7.0 was added to 3.4 nmol IMP 288. Four volumes of⁶⁸Ga eluate (380 MBq) were added and the mixture was heated to 95° C.for 20 min. Then 50 mM EDTA was added to a final concentration of 5 mMto complex the non-chelated ⁶⁸Ga³⁺. The ⁶⁸Ga-labeled IMP 288 peptide waspurified on a 1-mL Oasis HLB-cartridge (Waters, Milford, Mass.). Afterwashing the cartridge with water, the peptide was eluted with 25%ethanol. The procedure to label IMP 288 with ⁶⁸Ga was performed within45 minutes, with the preparations being ready for in vivo use.

Labeling of IMP 449

IMP 449 was labeled with ¹⁸F as described above. 555-740 MBq ¹⁸F (UV.Cyclotron VU, Amsterdam, The Netherlands) was eluted from a QMAcartridge with 0.4 M KHCO₃. The Al¹⁸F activity was added to a vialcontaining the peptide (230 μg) and ascorbic acid (10 mg). The mixturewas incubated at 100° C. for 15 mM. The reaction mixture was purified byRP-HPLC. After adding one volume of water, the peptide was purified on a1-mL Oasis HLB cartridge. After washing with water, the radiolabeledpeptide was eluted with 50% ethanol. ¹⁸F-IMP 449 was prepared within 60minutes, with the preparations being ready for in vivo use.

Radiochemical purity of ¹²⁵I-TF2, ¹¹¹In— and ⁶⁸Ga-IMP 288 and Al¹⁸F-IMP449 preparations used in the studies always exceeded 95%.

Animal Experiments

Experiments were performed in male nude BALB/c mice (6-8 weeks old),weighing 20-25 grams. Mice received a subcutaneous injection with 0.2 mLof a suspension of 1×10⁶ LS174T-cells, a CEA-expressing human coloncarcinoma cell line (American Type Culture Collection, Rockville, Md.,USA). Studies were initiated when the tumors reached a size of about0.1-0.3 g (10-14 days after tumor inoculation).

The interval between TF2 and IMP 288 injection was 16 hours, as thisperiod was sufficient to clear unbound TF2 from the circulation. In somestudies ¹²⁵I-TF2, (0.4 MBq) was co-injected with unlabeled TF2. IMP 288was labeled with either ¹¹¹In or ⁶⁸Ga. IMP 449 was labeled with ¹⁸F.Mice received TF2 and IMP 288 intravenously (0.2 mL). One hour after theinjection of ⁶⁸Ga-labeled peptide, and two hours after injection of¹⁸F-IMP 449, mice were euthanized by CO₂/O₂, and blood was obtained bycardiac puncture and tissues were dissected.

PET images were acquired with an Inveon animal PET/CT scanner (SiemensPreclinical Solutions, Knoxyille, Tenn.). PET emission scans wereacquired for 15 minutes, preceded by CT scans for anatomical reference(spatial resolution 113 μm, 80 kV, 500 μA, exposure time 300 msec).

After imaging, tumor and organs of interest were dissected, weighed andcounted in a gamma counter with appropriate energy windows for ¹²⁵I,¹¹¹In, ⁶⁸Ga or ¹⁸F. The percentage-injected dose per gram tissue (%ID/g) was calculated.

Results

Within 1 hour, pretargeted immunoPET resulted in high and specifictargeting of ⁶⁸Ga-IMP 288 in the tumor (10.7±3.6% ID/g), with very lowuptake in the normal tissues (e.g., tumor/blood 69.9±32.3), in aCEA-negative tumor (0.35±0.35% ID/g), and inflamed muscle (0.72±0.20%ID/g). Tumors that were not pretargeted with TF2 also had low ⁶⁸Ga-IMP288 uptake (0.20±0.03% ID/g). [¹⁸F]FDG accreted efficiently in the tumor(7.42±0.20% ID/g), but also in the inflamed muscle (4.07±1.13% ID/g) anda number of normal tissues, and thus pretargeted ⁶⁸Ga-IMP 288 providedbetter specificity and sensitivity. The corresponding PET/CT images ofmice that received ⁶⁸Ga-IMP 288 or ¹⁸F-labeled IMP 449 followingpretargeting with TF2 clearly showed the efficient targeting of theradiolabeled peptide in the subcutaneous LS174T tumor, while theinflamed muscle was not visualized. In contrast, with [¹⁸F]FDG the tumoras well as the inflammation was clearly delineated.

Dose Optimization

The effect of the TF2 bsMAb dose on tumor targeting of a fixed 0.01 nmol(15 ng) dose of IMP 288 was determined. Groups of five mice wereinjected intravenously with 0.10, 0.25, 0.50 or 1.0 nmol TF2 (16, 40, 80or 160 μg respectively), labeled with a trace amount of ¹²⁵I (0.4 MBq).One hour after injection of ¹¹¹In-IMP 288 (0.01 nmol, 0.4 MBq), thebiodistribution of the radiolabels was determined.

TF2 cleared rapidly from the blood and the normal tissues. Eighteenhours after injection the blood concentration was less than 0.45% ID/gat all TF2 doses tested. Targeting of TF2 in the tumor was 3.5% ID/g at2 h p.i. and independent of TF2 dose (data not shown). At all TF2 doses¹¹¹In-IMP 288 accumulated effectively in the tumor (not shown). Athigher TF2 doses enhanced uptake of ¹¹¹In-IMP 288 in the tumor wasobserved: at 1.0 nmol TF2 dose maximum targeting of IMP 288 was reached(26.2±3.8% ID/g). Thus at the 0.01 nmol peptide dose highest tumortargeting and tumor-to-blood ratios were reached at the highest TF2 doseof 1.0 nmol (TF2:IMP 288 molar ratio=100:1). Among the normal tissues,the kidneys had the highest uptake of ¹¹¹In IMP 288 (1.75±0.27% ID/g)and uptake in the kidneys was not affected by the TF2 dose (not shown).All other normal tissues had very low uptake, resulting in extremelyhigh tumor-to-nontumor ratios, exceeding 50:1 at all TF2 doses tested(not shown).

For PET imaging using ⁶⁸Ga-labeled IMP 288, a higher peptide dose isrequired, because a minimum activity of 5-10 MBq ⁶⁸Ga needs to beinjected per mouse if PET imaging is performed 1 h after injection. Thespecific activity of the ⁶⁸Ga-IMP 288 preparations was 50-125 MBq/nmolat the time of injection. Therefore, for PET imaging at least 0.1-0.25nmol of IMP 288 had to be administered. The same TF2:IMP 288 molarratios were tested at 0.1 nmol IMP 288 dose. LS174T tumors werepretargeted by injecting 1.0, 2.5, 5.0 or 10.0 nmol TF2 (160, 400, 800or 1600 μg). In contrast to the results at the lower peptide dose,¹¹¹In-IMP 288 uptake in the tumor was not affected by the TF2 doses (15%ID/g at all doses tested, data not shown). TF2 targeting in the tumor interms of % ID/g decreased at higher doses (3.21±0.61% ID/g versus1.16±0.27% ID/g at an injected dose of 1.0 nmol and 10.0 nmol,respectively) (data not shown). Kidney uptake was also independent ofthe bsMAb dose (2% ID/g). Based on these data we selected a bsMAb doseof 6.0 nmol for targeting 0.1-0.25 nmol of IMP 288 to the tumor.

PET Imaging

To demonstrate the effectiveness of pretargeted immunoPET imaging withTF2 and ⁶⁸Ga-IMP 288 to image CEA-expressing tumors, subcutaneous tumorswere induced in five mice. In the right flank an s.c. LS174T tumor wasinduced, while at the same time in the same mice 1×10⁶ SK-RC 52 cellswere inoculated in the left flank to induce a CEA-negative tumor.Fourteen days later, when tumors had a size of 0.1-0.2 g, the mice werepretargeted with 6.0 nmol ¹²⁵I-TF2 intravenously. After 16 hours themice received 5 MBq ⁶⁸Ga-IMP 288 (0.25 nmol, specific activity of 20MBq/nmol). A separate group of three mice received the same amount of⁶⁸Ga-IMP 288 alone, without pretargeting with TF2. PET/CT scans of themice were acquired 1 h after injection of the ⁶⁸Ga-IMP 288.

The biodistribution of ¹²⁵I-TF2 and ⁶⁸Ga-IMP 288 in the mice are shownin FIG. 6. Again high uptake of the bsMAb (2.17±0.50% ID/g) and peptide(10.7±3.6% ID/g) in the tumor was observed, with very low uptake in thenormal tissues (tumor-to-blood ratio: 64±22). Targeting of ⁶⁸Ga-IMP 288in the CEA-negative tumor SK-RC 52 was very low (0.35±0.35% ID/g).Likewise, tumors that were not pretargeted with TF2 had low uptake of⁶⁸Ga-IMP 288 (0.20±0.03% ID/g), indicating the specific accumulation ofIMP 288 in the CEA-expressing LS174T tumor.

The specific uptake of ⁶⁸Ga-IMP 288 in the CEA-expressing tumorpretargeted with TF2 was clearly visualized in a PET image acquired 1 hafter injection of the ⁶⁸Ga-labeled peptide (not shown). Uptake in thetumor was evaluated quantitatively by drawing regions of interest (ROI),using a 50% threshold of maximum intensity. A region in the abdomen wasused as background region. The tumor-to-background ratio in the image ofthe mouse that received TF2 and ⁶⁸Ga-IMP 288 was 38.2.

We then examined pretargeted immunoPET with [¹⁸F]FDG. In two groups offive mice a s.c. LS174T tumor was induced on the right hind leg and aninflammatory focus in the left thigh muscle was induced by intramuscularinjection of 50 μL turpentine (18). Three days after injection of theturpentine, one group of mice received 6.0 nmol TF2, followed 16 h laterby 5 MBq ⁶⁸Ga-IMP 288 (0.25 nmol). The other group received [¹⁸F]FDG (5MBq). Mice were fasted during 10 hours prior to the injection andanaesthetized and kept warm at 37° C. until euthanasia, 1 hpostinjection.

Uptake of ⁶⁸Ga-IMP 288 in the inflamed muscle was very low, while uptakein the tumor in the same animal was high (0.72±0.20% ID/g versus8.73±1.60% ID/g, p<0.05, FIG. 7). Uptake in the inflamed muscle was inthe same range as uptake in the lungs, liver and spleen (0.50±0.14%ID/g, 0.72±0.07% ID/g, 0.44±0.10% ID/g, respectively). Tumor-to-bloodratio of ⁶⁸Ga-IMP 288 in these mice was 69.9±32.3; inflamedmuscle-to-blood ratio was 5.9±2.9; tumor-to-inflamed muscle ratio was12.5±2.1. In the other group of mice ¹⁸F-FDG accreted efficiently in thetumor (7.42±0.20% ID/g, tumor-to-blood ratio 6.24±1.5, FIG. 4). ¹⁸F-FDGalso substantially accumulated in the inflamed muscle (4.07±1.13% ID/g),with inflamed muscle-to-blood ratio of 3.4±0.5, and tumor-to-inflamedmuscle ratio of 1.97±0.71.

The corresponding PET/CT image of a mouse that received ⁶⁸Ga-IMP 288,following pretargeting with TF2, clearly showed the efficient accretionof the radiolabeled peptide in the tumor, while the inflamed muscle wasnot visualized (FIG. 8). In contrast, on the images of the mice thatreceived ¹⁸F-FDG, the tumor as well as the inflammation was visible(FIG. 8). In the mice that received ⁶⁸Ga-IMP 288, the tumor-to-inflamedtissue ratio was 5.4; tumor-to-background ratio was 48; inflamedmuscle-to-background ratio was 8.9. [¹⁸F]FDG uptake had atumor-to-inflamed muscle ratio of 0.83; tumor-to-background ratio was2.4; inflamed muscle-to-background ratio was 2.9.

The pretargeted immunoPET imaging method was tested using theAl¹⁸F-labeled IMP 449. Five mice received 6.0 nmol TF2, followed 16 hlater by 5 MBq Al¹⁸F-IMP 449 (0.25 nmol). Three additional mice received5 MBq Al¹⁸F-IMP 449 without prior administration of TF2, while twocontrol mice were injected with [Al¹⁸F] (3 MBq). The results of thisexperiment are summarized in FIG. 9. Uptake of Al¹⁸F-IMP 449 in tumorspretargeted with TF2 was high (10.6±1.7% ID/g), whereas it was very lowin the non-pretargeted mice (0.45±0.38% ID/g). [Al¹⁸F] accumulated inthe bone (50.9±11.4% ID/g), while uptake of the radiolabeled IMP 449peptide in the bone was very low (0.54±0.2% ID/g), indicating that theAl¹⁸F-IMP 449 was stable in vivo. The biodistribution of Al¹⁸F-IMP 449in the TF2 pretargeted mice with s.c. LS174T tumors were highly similarto that of ⁶⁸Ga-IMP 288.

The PET-images of pretargeted immunoPET with Al¹⁸F-IMP 449 show the sameintensity in the tumor as those with ⁶⁸Ga-IMP 288, but the resolution ofthe ¹⁸F-images was superior to those of the ⁶⁸Ga-images (FIG. 10). Thetumor-to-background ratio of the Al¹⁸F-IMP 449 signal was 66.

Conclusions

The present study showed that pretargeted immunoPET with theanti-CEA×anti-HSG bispecific antibody TF2 in combination with a ⁶⁸Ga- or¹⁸F-labeled di-HSG-DOTA-peptide is a rapid and specific technique forPET imaging of CEA-expressing tumors.

Pretargeted immunoPET with TF2 in combination with ⁶⁸Ga-IMP 288 orAl¹⁸F-IMP 449 involves two intravenous administrations. An intervalbetween the infusion of the bsMAb and the radiolabeled peptide of 16 hwas used. After 16 h most of the TF2 had cleared from the blood (bloodconcentration <1% ID/g), preventing complexation of TF2 and IMP 288 inthe circulation.

For these studies the procedure to label IMP 288 with ⁶⁸Ga wasoptimized, resulting in a one-step labeling technique. We found thatpurification on a C18/HLB cartridge was needed to remove the ⁶⁸Gacolloid that is formed when the peptide was labeled at specificactivities exceeding 150 GBq/nmol at 95° C. If a preparation contains asmall percentage of colloid and is administered intravenously, the ⁶⁸Gacolloid accumulates in tissues of the mononuclear phagocyte system(liver, spleen, and bone marrow), deteriorating image quality. The⁶⁸Ga-labeled peptide could be rapidly purified on a C18-cartridge.Radiolabeling and purification for administration could be accomplishedwithin 45 minutes.

The half-life of ⁶⁸Ga matches with the kinetics of the IMP 288 peptidein the pretargeting system: maximum accretion in the tumor is reachedwithin 1 h. ⁶⁸Ga can be eluted twice a day form a ⁶⁸Ge/⁶⁸Ga generator,avoiding the need for an on-site cyclotron. However, the high energy ofthe positrons emitted by ⁶⁸Ga (1.9 MeV) limits the spatial resolution ofthe acquired images to 3 mm, while the intrinsic resolution of themicroPET system is as low as 1.5 mm.

¹⁸F, the most widely used radionuclide in PET, has an even morefavorable half-life for pretargeted PET imaging (t_(1/2)=110 min). TheNOTA-conjugated peptide IMP 449 was labeled with ¹⁸F, as describedabove. Like labeling with ⁶⁸Ga, it is a one-step procedure. Labelingyields as high as 50% were obtained. The biodistribution of Al¹⁸F-IMP449 was highly similar to that of ⁶⁸Ga-labeled IMP 288, suggesting thatwith this labeling method ¹⁸F is a residualizing radionuclide.

In contrast with FDG-PET, pretargeted radioimmunodetection is a tumorspecific imaging modality. Although a high sensitivity and specificityfor FDG-PET in detecting recurrent colorectal cancer lesions has beenreported in patients (Huebner et al., 2000, J Nucl Med 41:11277-89),FDG-PET images could lead to diagnostic dilemmas in discriminatingmalignant from benign lesions, as indicated by the high level oflabeling observed with inflammation. In contrast, the hightumor-to-background ratio and clear visualization of CEA-positive tumorsusing pretargeted immunoPET with TF2 ⁶⁸Ga- or ¹⁸F-labeled peptidessupports the use of the described methods for clinical imaging of cancerand other conditions. Apart from detecting metastases and discriminatingCEA-positive tumors from other lesions, pretargeted immunoPET could alsobe used to estimate radiation dose delivery to tumor and normal tissuesprior to pretargeted radioimmunotherapy. As TF2 is a humanized antibody,it has a low immunogenicity, opening the way for multiple imaging ortreatment cycles.

Example 23 Synthesis of Folic Acid NOTA Conjugate

Folic acid is activated as described (Wang et. al. Bioconjugate Chem.1996, 7, 56-62.) and conjugated to Boc-NH—CH₂—CH₂—NH₂. The conjugate ispurified by chromatography. The Boc group is then removed by treatmentwith TFA. The amino folate derivative is then mixed with p-SCN-Bn-NOTA(Macrocyclics) in a carbonate buffer. The product is then purified byHPLC. The folate-NOTA derivative is labeled with Al¹⁸F as described inthe preceding Examples and then HPLC purified. The ¹⁸F-labeled folate isinjected i.v. into a subject and successfully used to image thedistribution of folate receptors, for example in cancer or inflammatorydiseases (see, e.g., Ke et al., Advanced Drug Delivery Reviews,56:1143-60, 2004).

Example 24 Pretargeted PET Imaging in Humans

A patient (1.7 m² body surface area) with a suspected recurrent tumor isinjected with 17 mg of bispecific monoclonal antibody (bsMab). The bsMabis allowed to localize to the target and clear from the blood. The¹⁸F-labeled peptide (5-10 mCi on 5.7×10⁻⁹ mol) is injected when 99% ofthe bsMab has cleared from the blood. PET imaging shows the presence ofmicrometastatic tumors.

Example 25 Imaging of Angiogenesis Receptors by ¹⁸F Labeling

Labeled Arg-Gly-Asp (RGD) peptides have been used for imaging ofangiogenesis, for example in ischemic tissues, where α_(v)β₃ integrin isinvolved. (Jeong et al., J. Nucl. Med. 2008, Apr. 15 epub). RGD isconjugated to SCN-Bn-NOTA according to Jeong et al. (2008). [Al¹⁸F] isattached to the NOTA-derivatized RGD peptide as described above, bymixing aluminum stock solution with ¹⁸F and the derivatized RGD peptideand heating at 110° C. for 15 min, using an excess of peptide to drivethe labeling reaction towards completion. The ¹⁸F labeled RGD peptide isused for in vivo biodistribution and PET imaging as disclosed in Jeonget al. (2008). The [Al¹⁸F] conjugate of RGD-NOTA is taken up intoischemic tissues and provides PET imaging of angiogenesis.

Example 26 Carbohydrate Labeling

A NOTA thiosemicarbazide derivative is prepared by reacting thep-SCN-Bn-NOTA with hydrazine and then purifying the ligand by HPLC.[Al¹⁸F] is prepared as described in the preceding Examples and the[Al¹⁸F] is added to the NOTA thiosemicarbazide and heated for 15 min.Optionally the [Al¹⁸F] NOTA thiosemicarbazide complex is purified byHPLC. The [Al¹⁸F] NOTA thiosemicarbazide is conjugated to oxidizedcarbohydrates by known methods. The ¹⁸F-labeled carbohydrate issuccessfully used for imaging studies using PET scanning.

Example 27 Effect of Organic Solvents on F-18 Labeling

The affinity of chelating moieties such as NETA and NOTA for aluminum ismuch higher than the affinity of aluminum for ¹⁸F. The affinity of Alfor ¹⁸F is affected by factors such as the ionic strength of thesolution, since the presence of other counter-ions tends to shield thepositively charged aluminum and negatively charged fluoride ions fromeach other and therefore to decrease the strength of ionic binding.Therefore low ionic strength medium should increase the effectivebinding of Al and ¹⁸F.

An initial study adding ethanol to the ¹⁸F reaction was found toincrease the yield of radiolabeled peptide. IMP 461 was prepared asdescribed above.

TABLE 8 ¹⁸F labeling of IMP 461 in ethanol # 2 mM AlCl₃ F-18 2 mM IMP461 Solvent Yield* 1 10 μL 741 μCi 20 μL EtOH 60 μL 64.9% 2 10 μL 739μCi 20 μL H₂O 60 μL 21.4% 3 10 μL 747 μCi 20 μL EtOH 60 μL 46.7% 4  5 μL947 μCi 10 μL EtOH 60 μL 43.2% *Yield after HLB column purification, Rxn# 1, 2 and 4 were heated to 101° C. for 5 minutes, Rxn # 3 was heatedfor 1 minute in a microwave oven.

Preliminary results showed that addition of ethanol to the reactionmixture more than doubled the yield of ¹⁸F-labeled peptide. Table 8 alsoshows that microwave irradiation can be used in place of heating topromote incorporation of [Al¹⁸F] into the chelating moiety of IMP 461.Sixty seconds of microwave radiation (#3) appeared to be slightly less(18%) effective than heating to 101° C. for 5 minutes (#1).

The effect of additional solvents on 19F labeling of peptides wasexamined. In each case, the concentration of reactants was the same andonly the solvent varied. Reaction conditions included mixing 25 μLNa¹⁹F+20 μL AlCl₃+20 μL IMP-461+60 μL solvent, followed by heating at101° C. for 5 min. Table 9 shows that the presence of a solvent doesimprove the yields of [Al¹⁹F] IMP-461 (IMP 473) considerably.

TABLE 9 ¹⁹F labeling of IMP 461 in various solvents Solvent H₂O MeOHEtOH CH₃CN Al-IMP-461 2.97 3.03 2.13 1.54 IMP-465 52.46 34.19 31.5824.58 IMP-473 14.99 30.96 33.00 37.48 IMP-473 15.96 31.81 33.29 36.40IMP-461 13.63 — — — Solvent IPA Acetone THF Dioxane Al-IMP-461 2.02 2.052.20 16.67 IMP-465 32.11 28.47 34.76 10.35 IMP-473 27.31 34.35 29.3827.09 IMP-473 27.97 35.13 29.28 11.62 IMP-461 10.58 — 4.37 34.27 SolventDMF DMSO t_(R) (min) Al-IMP-461 — — 9.739 IMP-465 19.97 37.03 10.138IMP-473 27.77 31.67 11.729 IMP-473 27.34 31.29 11.952 IMP-461 — — 12.535[A1¹⁹F] IMP 461 = IMP 473

Example 28 Elution of ¹⁸F with Bicarbonate

¹⁸F, 10.43 mCi, was received in 2 mL in a syringe. The solution waspassed through a SEP-PAK® Light, WATERS® ACCELL™ Plus QMA Cartridge. Thecolumn was then washed with 5 mL of DI water. The ¹⁸F was eluted with0.4 M KHCO₃ in fractions as shown in Table 10 below.

TABLE 10 Elution of QMA Cartridge with KHCO₃ Vial Vol. Acetic acid μLVol. 0.4 M KHCO₃ μL Activity mCi 1 7.5 150 0.0208 2 10 200 7.06 3 5 1001.653 4 25 500 0.548

The effects of the amount of additional solvent (CH₃CN) on ¹⁸F labelingof IMP-461 was examined. In each case, the concentration of reactantswas the same and only the amount of solvent varied. Reaction conditionsincluded mixing 10 μL AlCl₃+20 μL ¹⁸F+20 μL IMP-461+CH₃CN followed byheating at 101° C. for 5 min. Table 11 shows that following an initialimprovement the labeling efficiency decreases in the presence of excesssolvent.

TABLE 11 ¹⁸F labeling of IMP 461 using varying amounts of CH₃CN RCY %CH3CN (μL) F-18 mCi t_(R) 2.70 min (%) t_(R) 8.70 min (%) (HLB) 0 0.64213.48 86.52 50.7 100 0.645 1.55 98.45 81.8* 200 0.642 2.85 97.15 80.8400 0.645 14.51 85.49 57.8 *Aqueous wash contains labeled peptide. RCY =radiochemical yield after HLB purification

Example 29 High Dose Radiolabeling of IMP 461

¹⁸F, 163 mCi, was received in 2 mL in a syringe. The solution was passedthrough a SEP-PAK® Light, WATERS® ACCELL™ Plus QMA Cartridge. The columnwas then washed with 5 mL of DI water. The ¹⁸F was eluted with 0.4 MK₂CO₃ in fractions as shown in Table 12.

TABLE 12 High Dose Labeling Vial Vol. Acetic acid μL Vol. 0.4 M K₂CO₃ μLActivity mCi 1 18.5 185 5.59 2 5 50 35.8 3 5 50 59.9 4 5 50 20.5 5 5 505.58 6 50 500 4.21

An aluminum chloride solution (10 μL, 2 mM in pH 4, 2 mM NaOAc) wasadded to vial number 3 from Table 12. The peptide (20 μL, 2 mM in pH 4,2 mM NaOAc) was added to the vial followed by the addition of 170 μL ofCH₃CN. The solution was heated for 10 min at 103° C. the diluted with 6mL of water. The solution was pulled into a 10 mL syringe and injectedonto two WATERS® HLB Plus Cartridges arranged in tandem. The cartridgeswere washed with 8 mL water. The radiolabeled peptide Al¹⁸F IMP 461 wasthen eluted with 10 mL 1:1 EtOH/H₂O, 30.3 mCi, 63.5% yield, specificactivity 750 Ci/mmol. The labeled peptide was free of unbound ¹⁸F byHPLC. The total reaction and purification time was 20 min.

Example 30 Preparation of ¹⁹F Labeled Peptides

Products containing ²⁷A1 and/or ¹⁹F are useful for certain applicationslike NMR imaging. An improved method for preparing [Al¹⁹F] labeledcompounds was developed. IMP 461 was prepared as described above andlabeled with ¹⁹F. Reacting IMP 461 with AlCl₃+NaF resulted in theformation of three products (not shown). However, by reacting IMP 461with AlF₃.6H₂O we obtained a higher yield of [Al¹⁹F]IMP 461.

Synthesis of IMP 473

([Al¹⁹F] IMP 461) To (14.1 mg, 10.90 μmol) IMP 461 in 2 mL NaOAc (2 mM,pH 4.18) solution added (4.51 mg, 32.68 mmol) AlF₃.6H₂O and 500 μLethanol. The pH of the solution to adjusted to 4.46 using 3 μL1N NaOHand heated in a boiling water bath for 30 minutes. The crude reactionmixture was purified by preparative RP-HPLC to yield 4.8 mg (32.9%) ofIMP 473. HRMS (ESI-TOF) MH⁺expected 1337.6341; found 1337.6332

These results demonstrate that ¹⁹F labeled molecules may be prepared byforming metal-¹⁹F complexes and binding the metal-¹⁹F to a chelatingmoiety, as discussed above for ¹⁸F labeling. The instant Example showsthat a targeting peptide of use for pretargeting detection, diagnosisand/or imaging may be prepared using the instant methods.

Example 31 Synthesis and Labeling of IMP 479, IMP 485 and IMP 487

The structures of additional peptides IMP 479 (SEQ ID NO:35), IMP 485(SEQ ID NO:36) and IMP 487 (SEQ ID NO:37) designed for ¹⁸F-labeling areshown in FIG. 11 to FIG. 13. IMP 485 is shown in FIG. 12. IMP 485 wasmade on Sieber Amide resin by adding the following amino acids to theresin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc wascleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, (tent-Butyl)₂NODA-MPAA (methyl phenyl acetic acid). Thepeptide was then cleaved from the resin and purified by RP-HPLC to yield44.8 mg of IMP 485.

Synthesis of Bis-t-butyl-NODA-MPAA: NO₂AtBu-MPAA for IMP 485 Synthesis

To a solution of 4-(bromomethyl)phenyl acetic acid (Aldrich 310417)(0.5925 g, 2.59 mmol) in CH₃CN (anhydrous) (50 mL) at 0° C. was addeddropwise over 1 h a solution of NO₂AtBu (1.0087 g, 2.82 mmol) in CH₃CN(50 mL). After 4 h anhydrous K₂CO₃ (0.1008 g, 0.729 mmol) was added tothe reaction mixture and allowed to stir at room temperature overnight.Solvent was evaporated and the crude mixture was purified by preparativeRP-HPLC to yield a white solid (0.7132 g, 54.5%).

Although this is a one step synthesis, yields were low due toesterification of the product by 4-(bromomethyl)phenylacetic acid.Alkylation of NO2AtBu using methyl(4-bromomethyl)phenylacetate wasemployed to prevent esterification (FIG. 14).

¹⁸F Labeling

For ¹⁸F labeling studies in water, to 40 nmol of IMP-479/485/487(formulated using trehalose+ascorbic acid+AlCl₃) was added 250 μL F-18solution [˜ 919-1112 μCi of F-18] and heated to 101° C. for 15 minutes.In ethanol, to 40 nmol of IMP-479/485/487 (formulated usingtrehalose+ascorbic acid+AlCl₃) was added 250 μL F-18 solution[1.248-1.693 mCi of F-18], 100 μL EtOH and heated to 101° C. for 15minutes. An exemplary experiment showing labeling of different peptidesis shown in Table 13. With minimal optimization, radiolabeling of IMP485 has been observed with up to an 88% yield and a specific activity of2,500 Ci/mmol. At this specific activity, HPLC purification of theradiolabeled peptide is not required for in vivo PET imaging using theradiolabeled peptide.

TABLE 13 Labeling of IMP 479, IMP 485 and IMP 487 Isolated yields afterHLB purification IMP # H₂O EtOH IMP-479 44.0% 57.5% IMP 485 74.4% 79.7%IMP-487 63.6% 81.6%

Stability in Serum

A kit containing 40 nmol of IMP 485 or IMP 487, 20 nmol AlCl₃, 0.1 mgascorbic acid and 0.1 g trehalose adjusted to pH 3.9 was reconstitutedwith purified ¹⁸F in 200 μL saline and heated 106° C. for 15 min. Thereaction mixture was then diluted with 800 μL water and placed on an HLBcolumn. After washing, the column was eluted with 2×200 μL 1:1 EtOH/H₂Oto obtain the purified ¹⁸F-IMP 485 in 64.6% isolated yield. Theradiolabeled peptide in 50 μL was mixed with 250 μL of fresh human serumin a vial and incubated at 37° C.

Both radiolabeled peptides were stable at 37° C. in fresh human serumover the four hours tested (not shown).

Effect of Bulking Agents on Yield of Lyophilized Peptide

An experiment was performed to compare yield using IMP 485 kits (40nmol) with different bulking agents labeled with 2 mCi of F-18 (from thesame batch of F-18) in 200 microliters of saline. The bulking agentswere introduced at a concentration of 5% by weight in water with a doseof 200 microliters/vial. We tested sorbitol, trehalose, sucrose,mannitol and glycine as bulking agents. Results are shown in Table 14

TABLE 14 Effects of Bulking Agents on Radiolabeling Yield Bulking AgentActivity mCi Yield % Sorbitol 2.17 82.9 Glycine 2.17 41.5 Mannitol 2.1181.8 Sucrose 2.11 66.1 Trehalose 2.10 81.3

Sorbitol, mannitol and trehalose all gave radiolabeled product in thesame yield. The mannitol kit and the trehalose kit both formed nicecakes. The sucrose kit and the glycine kit both had significantly loweryields. We also recently tested 2-hydroxypropyl-beta-cyclodextrin as abulking agent and obtained a 58% yield for the 40 nmol kit. We havefound that radiolabeling is very pH sensitive and needs to be tuned tothe ligand and possibly even to the peptide+the ligand. In the case ofIMP 485 the optimal pH is pH 4.0±0.2 whereas the optimal pH for IMP 467was pH 4.5±0.5. In both cases the yields drop off rapidly outside theideal pH zone.

Time Course of Labeling

The time course for labeling of IMP 485 was examined. To 40 nmol of IMP485 (formulated using trehalose+AlCl₃ (20 nmol)+ascorbic acid) was added˜200-250 μL F-18 solution (0.9% saline) and heated to 104° C. for 5 to15 minutes. The results for labeling yield were: 5 min (28.9%), 10 min(57.9%), 15 min (83.7%) and 30 min (88.9%). Thus, the time course forlabeling was approximately 15 minutes.

Biodistribution of IMP 485 Alone

The biodistribution of IMP 485 in the absence of any pretargetingantibody was examined in female Taconic nude mice (10 week old) bearingsmall or no BXPC3 pancreatic cancer xenografts. The mice were injectedi.v. with ¹⁸F-IMP 485, (340 μCi, 2.29×10⁻⁹ mol, 100 μL in saline). Themice, 6 per time point, were necropsied at 30 min and 90 min postinjection. In the absence of pretargeting antibody a low level ofaccumulation was seen in tumor and most normal tissues. The substantialmajority of radiolabel was found in the bladder and to a lesser extentin kidney. Most of the activity was cleared before the 90 min timepoint.

Pretargeting of IMP 485 with TF2 DNL Targeting Molecule

¹⁸F-IMP 485 Radiolabeling ¹⁸F (218 mCi) was purified to isolate 145.9mCi. The purified ¹⁸F (135 mCi) was added to a lyophilized vialcontaining 40 nmol of pre-complexed Al-IMP 485. The reaction vial washeated at 110° C. for 17 min. Water (0.8 mL) was added to the reactionmixture before HLB purification. The product (22 mCi) was eluted with0.6 mL of water:ethanol (1:1) mixture into a vial containing lyophilizedascorbic acid. The product was diluted with saline. The ¹⁸F—Al IMP 485specific activity used for injection was 550 Ci/mmol.

Biodistribution of ¹⁸F—Al IMP 485 Alone

Mice bearing sc LS174T xenografts were injected with ¹⁸F—Al IMP 485 (28μCi, 5.2×10⁻¹¹ mol, 100 μL. Mice were necropsied at 1 and 3 h postinjection, 6 mice per time point.

Biodistribution of TF2+¹⁸F—Al IMP 485 With Pretargeting at 20:1 bsMAb toPeptide Ratio

Mice bearing sc LS174T xenografts were injected with TF2 (163.2 μg,1.03×10⁻⁹ mol, iv) and allowed 16.3 h for clearance before injecting¹⁸F—Al IMP 485 (28 μCi, 5.2×10⁻¹¹ mol, 100 μL iv). Mice were necropsiedat 1 and 3 h post injection, 7 mice per time point.

Urine Stability

Ten mice bearing s.c. Capan-1 xenografts were injected with ¹⁸F—Al-IMP485 (400 μCi, in saline, 100 μL). Urine was collected from 3 mice at 55min post injection. The urine samples were analyzed by reverse phase andSE HPLC. Stability of the radiolabeled IMP 485 in urine was observed.

TABLE 15 ¹⁸F-IMP 485 Alone at 1 h post injection: STD STD STD STD Tissuen Weight WT % ID/g % ID/g % ID/org % ID/org T/NT T/NT Tumor 6 0.2350.147 0.316 0.114 0.081 0.063 1.0 0.0 Liver 6 1.251 0.139 0.176 0.0320.220 0.043 1.8 0.4 Spleen 6 0.085 0.019 0.210 0.181 0.018 0.017 1.9 0.9Kidney 6 0.149 0.013 3.328 0.556 0.499 0.119 0.1 0.0 Lung 6 0.141 0.0390.238 0.048 0.033 0.010 1.3 0.3 Blood 6 0.222 0.006 0.165 0.062 0.2680.101 2.0 0.4 Stomach 6 0.478 0.083 0.126 0.110 0.057 0.045 3.5 1.6 SmInt. 6 0.896 0.098 0.396 0.128 0.353 0.110 0.8 0.3 Lg Int. 6 0.504 0.0560.081 0.019 0.041 0.010 3.9 0.9 Muscle 6 0.103 0.029 0.114 0.079 0.0110.008 4.1 2.5 Scapula 6 0.057 0.015 0.107 0.019 0.006 0.001 2.9 0.7

TABLE 16 ¹⁸F-IMP 485 Alone at 3 h post injection: STD STD STD STD Tissuen Weight WT % ID/g % ID/g % ID/org % ID/org T/NT T/NT Tumor 6 0.2650.126 0.088 0.020 0.022 0.011 1.0 0.0 Liver 6 1.219 0.091 0.095 0.0470.114 0.056 13.6 31.4 Spleen 6 0.091 0.015 0.065 0.009 0.006 0.001 1.40.2 Kidney 6 0.154 0.013 2.265 0.287 0.345 0.028 0.0 0.0 Lung 6 0.1420.008 0.073 0.019 0.010 0.003 1.3 0.6 Blood 6 0.236 0.019 0.008 0.0050.013 0.007 21.0 27.9 Stomach 6 0.379 0.054 0.041 0.017 0.016 0.008 2.51.0 Sm. Int. 6 0.870 0.042 0.137 0.031 0.119 0.029 0.7 0.3 Lg. Int. 60.557 0.101 0.713 0.215 0.408 0.194 0.1 0.0 Muscle 6 0.134 0.038 0.0130.007 0.002 0.001 203.9 486.6 Scapula 6 0.074 0.009 0.079 0.026 0.0060.002 1.2 0.6

TABLE 17 TF2 + ¹⁸F-IMP 485, at 1 h post peptide injection: STD STD STDSTD Tissue n Weight WT % ID/g % ID/g % ID/org % ID/org T/NT T/NT Tumor 70.291 0.134 28.089 4.545 8.025 3.357 1 0 Liver 7 1.261 0.169 0.237 0.0370.295 0.033 123 38 Spleen 7 0.081 0.013 0.254 0.108 0.020 0.008 139 87Kidney 7 0.140 0.018 3.193 0.730 0.444 0.098 9 4 Lung 7 0.143 0.0140.535 0.147 0.075 0.018 57 22 Blood 7 0.205 0.029 0.278 0.071 0.4560.129 110 43 Stomach 7 0.473 0.106 0.534 1.175 0.265 0.598 381 318 Sm.Int. 7 0.877 0.094 0.686 0.876 0.586 0.725 75 39 Lg. Int. 7 0.531 0.0680.104 0.028 0.055 0.015 291 121 Muscle 7 0.090 0.014 0.136 0.102 0.0120.009 348 274 Scapula 6 0.189 0.029 0.500 0.445 0.095 0.092 120 108

TABLE 18 TF2 + ¹⁸F-IMP 485, at 3 h post peptide injection: STD STD STDSTD Tissue n Weight WT % ID/g % ID/g % ID/org ID/org T/NT T/NT Tumor 70.320 0.249 26.518 5.971 8.127 5.181 1 0 Liver 7 1.261 0.048 0.142 0.0190.178 0.025 189 43 Spleen 7 0.079 0.012 0.138 0.031 0.011 0.002 195 41Kidney 7 0.144 0.012 2.223 0.221 0.319 0.043 12 3 Lung 7 0.145 0.0140.244 0.056 0.035 0.005 111 24 Blood 7 0.229 0.014 0.023 0.008 0.0370.012 1240 490 Stomach 7 0.430 0.069 0.025 0.017 0.010 0.005 1389 850Sm. Int. 7 0.818 0.094 0.071 0.029 0.059 0.028 438 207 Lg. Int. 7 0.5860.101 0.353 0.160 0.206 0.103 86 33 Muscle 7 0.094 0.014 0.025 0.0060.002 0.001 1129 451 Scapula 7 0.140 0.030 0.058 0.018 0.008 0.002 502193

Conclusions

The IMP 485 labels as well as or better than IMP 467, with equivalentstability in serum. However, IMP 485 is much easier to synthesize thanIMP 467. Preliminary studies have shown that ¹⁸F labeling of lyophilizedIMP 485 works as well as non-lyophilized peptide (data not shown). Thepresence of alkyl or aryl groups in the linker joining the chelatingmoiety to the rest of the peptide was examined. The presence of arylgroups in the linker appears to increase the radiolabeling yieldrelative to the presence of alkyl groups in the linker.

Biodistribution of IMP 485 in the presence or absence of pretargetingantibody resembles that observed with IMP 467. In the absence ofpretargeting antibody, distribution of radiolabeled peptide in tumor andmost normal tissues is low and the peptide is removed from circulationby kidney excretion. In the presence of the TF2 antibody, radiolabeledIMP 485 is found primarily in the tumor, with little distribution tonormal tissues. Kidney radiolabeling is substantially decreased in thepresence of the pretargeting antibody. We conclude that IMP 485 andother peptides with aryl groups in the linker are highly suitable forPET imaging with ¹⁸F labeling.

Example 32 Kit Formulation of IMP 485 for Imaging

Reagents List

Reagents were obtained from the following sources: Acetic acid (JT Baker6903-05 or 9522-02), Sodium hydroxide (Aldrich semiconductor grade99.99% 30, 657-6), α,α-Trehalose (JT Baker 4226-04), Aluminum chloridehexahydrate (Aldrich 99% 237078), Ascorbic acid (Aldrich 25, 556-4).

Acetate Buffer 2 mM

Acetic acid, 22.9 μL (4.0×10⁻⁴ mol) was diluted with 200 mL water andneutralized with 6 N NaOH 15 μL) to adjust the solution to pH 4.22.

Aluminum Solution 2 mM

Aluminum hexahydrate, 0.0225 g (9.32×10⁵ mol) was dissolved in 47 mL DIwater.

α,α-Trehalose Solution

α,α-Trehalose, 4.004 g was dissolved in 40 mL DI water to make a 10%solution.

Peptide Solution, IMP 485 2 mM

The peptide IMP 485 (0.0020 g, 1.52 mol) was dissolved in 762 μL of 2 mMacetate buffer. The pH was 2.48 (the peptide was lyophilized as the TFAsalt). The pH of the peptide solution was adjusted to pH 4.56 by theaddition of 4.1 μL of 1M NaOH.

Ascorbic Acid Solution 5 mg/mL

Ascorbic acid, 0.0262 g (1.49×10⁴ mol) was dissolved in 5.24 mL DIwater.

Formulation of Peptide Kit

The peptide, 20 μL (40 nmol) was mixed with 12 μL (24 nmol) of Al, 100μL of trehalose, 20 μL (0.1 mg) ascorbic acid and 900 μL of DI water ina 3 mL lyophilization vial. The final pH of the solution should be ˜pH4.0. The vial was frozen, lyophilized and sealed under vacuum. In thisformulation, the peptide is loaded with aluminum prior to binding ¹⁸F.

Ten and 20 nmol kits have also been made. These kits are made the sameas the 40 nmol kits keeping the peptide to Al³⁺ratio of 1 peptide to 0.6Al³⁺but formulated in 2 mL vials with a total fill of 0.5 mL.

Purification of ¹⁸F

The crude ¹⁸F was received in 2 mL of DI water in a syringe. The syringewas placed on a syringe pump and the liquid pushed through a Waters CMcartridge followed by a QMA cartridge. Both cartridges were washed with10 mL DI water. A sterile disposable three way valve between the twocartridges was switched and 1 mL commercial sterile saline was pushedthrough the QMA cartridge in 200 μL fractions. The second fractionusually contains ˜80% of the ¹⁸F regardless of the amount of ¹⁸F applied(10-300 mCi loads were tested).

We alternatively use commercial ¹⁸F in saline, which has been purifiedon a QMA cartridge. This is a concentrated version of the commercialbone imaging agent so it is readily available and used in humans. Theactivity is supplied in 200 μL in a 0.5 mL tuberculin syringe.

Radiolabeling

The peptide was radiolabeled by adding ¹⁸F in 200 μL saline to thelyophilized peptide in a crimp sealed vial and then heating the solutionto 90-105° C. for 15 min. The peptide was purified by adding 800 mL ofDI water in a 1 mL syringe to the reaction vial, removing the liquidwith the 1 mL syringe and applying the liquid to a Waters HLB column(lcc, 30 mg). The HLB column was placed on a crimp sealed 5 mL vial andthe liquid was drawn into the vial under vacuum supplied by a remote(using a sterile disposable line) 10 mL syringe. The reaction vial waswashed with two one mL aliquots of DI water, which were also drawnthrough the column. The column was then washed with 1 mL more of DIwater. The column was then moved to a vial containing bufferedlyophilized ascorbic acid (˜pH 5.5, 15 mg). The radiolabeled product waseluted with three 200 μL portions of 1:1 EtOH/DI water. The yield wasdetermined by measuring the activity on the HLB cartridge, in thereaction vial, in the water wash and in the product vial to get thepercent yield.

Adding ethanol to the radiolabeling reaction can increase the labelingyield. A 20 nmol kit can be reconstituted with a mixture of 200 μL F-18in saline and 200 μL ethanol. The solution is then heated to 100-110° C.in the crimp sealed vial for 15 min. After heating, the reaction isdiluted with 3 mL water before purification on a 3 cc (60 mg) HLBextraction cartridge. The peptide can be labeled in good yield and up to4,100 Ci/mmol specific activity using this method.

The yield for this kit and label as described was 80-90% when labeledwith 1.0 mCi of ¹⁸F. When higher doses of ¹⁸F (˜100 mCi) were used theyield dropped. However if ethanol is added to the labeling mixture theyield goes up. If the peptides are diluted too much in saline the yieldswill drop. The labeling is also very sensitive to pH. For our peptidewith this ligand we have found that the optimal pH for the finalformulation was pH 4.0±0.2.

The purified radiolabeled peptide in 50 L 1:1 EtOH/H₂O was mixed with150 L of human serum and placed in the HPLC autosampler heated to 37° C.and analyzed by RP-HPLC. No detectable ¹⁸F above background at the voidvolume was observed even after 4 h.

TABLE 19 IMP 485 Labeling Activity nmol of Specific of Volume VolumeActivity isolated Activity Vial #/ Pep- Saline EtOH at start product %Ci/ Peptide tide μL μL mCi mCi Yield mmol 1. IMP485 10 100 0 20.0 7.1062 2. IMP485 10 50 50 19.4 9.43 78 3. IMP485 10 200 0 19.07 5.05 38 4.IMP485 20 100 100 37.3 22.3 80 5. IMP485 10 100 0 45.7 16.2 42 6. IMP48520 200 200 175.6 82.7 58 4135

Synthesis and Radiolabeling of IMP 486 (Al-OHIMP485)

IMP 485 (21.5 mg, 0.016 mmol) was dissolved in 1 mL of 2 mM NaOAc, pH4.4 and treated with AlCl₃.6H₂O (13.2 mg, 0.055 mmol). The pH wasadjusted to 4.5-5.0 and the reaction mixture was refluxed for 15minutes. The crude mixture was purified by preparative RP-HPLC to yielda white solid (11.8 mg).

The pre-filled Al-NOTA complex (IMP 486) was also radiolabeled inexcellent yield after formulating into lyophilized kits. The labelingyields with IMP 486 were as good as or better than IMP 485 kits (Table19) when labeled in saline. This high efficiency of radiolabeling withchelator preloaded with aluminum was not observed with any of the otherAl-NOTA complexes tested (data not shown). The equivalency of labelingin saline and in 1:1 ethanol/water the labeling yields was also notobserved with other chelating moieties (not shown).

TABLE 20 IMP 486 Labeling Activity of Specific Peptide Volume VolumeActivity at isolated % Activity 20 nmol Saline EtOH start mCi productmCi Yield Ci/mmol IMP486 100 μL  0 46 28 76 2800 IMP485 100 μL 100 μL 4125 83 IMP486 100 μL 100 μL 43 22 81 IMP486 200 μL  0 42 18 73

Effect of Bulking Agents

An experiment was performed to compare yield using IMP 485 kits (40nmol) with different bulking agents. The peptide was labeled with 2 mCiof F-18 from the same batch of F-18 in 200 microliters saline. Thebulking agents were introduced in water at a concentration of 5% byweight, with a dose of 200 microliters/vial. We tested sorbitol,trehalose, sucrose, mannitol and glycine as bulking agents. Results areshown in Table 20

TABLE 21 Effects of Bulking Agents on Radiolabeling Yield Bulking AgentActivity mCi Yield % Sorbitol 2.17 82.9 Glycine 2.17 41.5 Mannitol 2.1181.8 Sucrose 2.11 66.1 Trehalose 2.10 81.3

Sorbitol, mannitol and trehalose all gave radiolabeled product in thesame yield. The sucrose kit and the glycine kit both had significantlylower yields. Trehalose was formulated into IMP 485 kits atconcentrations ranging from 2.5% to 50% by weight when reconstituted in200 L. The same radiolabeling yield, ˜83%, was obtained for allconcentrations, indicating that the ¹⁸F-radiolabeling of IMP 485 was notsensitive to the concentration of the trehalose bulking agent. IMP 485kits were formulated and stored at 2-8° C. under nitrogen for up tothree days before lyophilization to assess the impact of lyophilizationdelays on the radiolabeling. The radiolabeling experiments indicatedthat yields were all ˜80% at time zero, and with 1, 2, and 3 days ofdelay before lyophilization.

Effect of pH on Radiolabeling

The effect of pH on radiolabeling of IMP 485 is shown in Table 21. Theefficiency of labeling was pH sensitive and decreased at either higheror lower pH relative to the optimal pH of about 4.0.

TABLE 22 Effect of pH on IMP 485 Radiolabeling Efficiency. pH Yield %3.27 33 3.53 61 3.84 85 3.99 88 4.21 89 4.49 80 5.07 14

Biodistribution

Biodistribution studies were performed in Taconic nude mice bearingsubcutaneous LS174T tumor xenografts.

Al¹⁸F-IMP 485 alone:

Mice bearing sc LS174T xenografts were injected with Al¹⁸F-IMP 485 (28Ci, 5.2×10⁻¹¹ mol, 100 L, iv). Mice were necropsied at 1 and 3 h postinjection, 6 mice per time point. TF2+Al¹⁸F-IMP 485 Pretargeting at 20:1bsMab to peptide ratio: Mice bearing sc LS174T xenografts were injectedwith TF2 (163.2 g, 1.03×10⁹ mol, iv) and allowed 16.3 h for clearancebefore injecting Al¹⁸F-IMP 485 (28 Ci, 5.2×10¹¹ mol, 100 L, iv). Micewere necropsied at 1 and 3 h post injection, 7 mice per time point.

TABLE 23 Biodistribution of TF2 pretargeted Al¹⁸F[IMP 485] or Al¹⁸F[IMP485] alone at 1 and 3 h after peptide injection in nude mice bearingLS174T human colonic cancer xenografts. Percent-injected dose per gramtissue (mean ± SD; N = 7) TF2 pregargeted Al¹⁸F[IMP485] Al¹⁸F[IMP485]alone Tissue 1 h 3 h 1 h 3 h Tumor 28.09 ± 4.55  26.52 ± 5.97  0.32 ±0.1  10.09 ± 0.02  Liver 0.24 ± 0.04 0.14 ± 0.02 0.18 ± 0.03 0.10 ± 0.05Spleen 0.25 ± 0.11 0.25 ± 0.11 0.21 ± 0.18 0.07 ± 0.01 Kidney 3.19 ±0.73 2.22 ± 0.22 3.33 ± 0.56 2.27 ± 0.29 Lung 0.54 ± 0.15 0.24 ± 0.060.24 ± 0.05 0.07 ± 0.02 Blood 0.28 ± 0.07 0.02 ± 0.01 0.17 ± 0.06 0.09 ±0.01 Stomach 0.53 ± 1.18 0.03 ± 0.02 0.13 ± 0.11 0.04 ± 0.02 Sm. Int.0.69 ± 0.88 0.07 ± 0.03 0.40 ± 0.13 0.14 ± 0.03 Lg. Int. 0.10 ± 0.030.35 ± 0.16 0.08 ± 0.02 0.71 ± 0.22 Muscle 0.14 ± 0.10 0.03 ± 0.01 0.11± 0.08 0.01 ± 0.01 Scapula  0.5 ± 0.45 0.06 ± 0.02 0.11 ± 0.02 0.03 ±0.01

Synthesis of IMP 492 (Al¹⁹FIMP485)

IMP485 (16.5 mg, 0.013 mmol) was dissolved in 1 mL of 2 mM NaOAc, pH4.43, 0.5 mL ethanol and treated with AlF₃.3H₂O (2.5 mg, 0.018 mmol).The pH was adjusted to 4.5-5.0 and the reaction mixture was refluxed for15 minutes. On cooling the pH was once again raised to 4.5-5.0 and thereaction mixture refluxed for 15 minutes. The crude was purified bypreparative RP-HPLC to yield a white solid (10.3 mg).

Synthesis of IMP 490

(SEQ ID NO: 38) NODA-MPAA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl

The peptide was synthesized on threoninol resin with the amino acidsadded in the following order: Fmoc-Cys(Trt)-OH, Fmoc-Thr(But)-OH,Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH,Fmoc-D-Phe-OH and (tBu)₂NODA-MPAA. The peptide was then cleaved andpurified by preparative RP-HPLC. The peptide was cyclized by treatmentof the bis-thiol peptide with DMSO.

Synthesis of IMP 491 (Al¹⁹FIMP490)

The Al¹⁹FIMP490 was prepared as described above (IMP492) to produce thedesired peptide after HPLC purification.

Synthesis of IMP 493

(SEQ ID NO: 39) NODA-MPAA-(PEG)₃-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂

The peptide was synthesized on Sieber amide resin with the amino acidsadded in the following order: Fmoc-Met-OH, Fmoc-Leu-OH,Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH,Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-(PEG)₃-COOH and(tBu)₂NODA-MPAA. The peptide was then cleaved and purified bypreparative RP-HPLC.

Synthesis of IMP 494 (Al¹⁹FIMP493)

The Al¹⁹FIMP493 was prepared as described above (1 MP492) to produce thedesired peptide after HPLC purification.

Example 33 Other Prosthetic Group Labeling Methods Using Al¹⁸F or Al¹⁹F

In certain embodiments, the aluminum fluoride labeling method may beperformed using prosthetic group labeling methods for molecules that aresensitive to heat. Prosthetic group conjugation may be carried out atlower temperatures for heat-sensitive molecules.

The prosthetic group NOTA is labeled with ¹⁸F or ¹⁹F as described aboveand then it is attached to the targeting molecule. In one non-limitingexample, this is performed with an aldehyde NOTA that is then attachedto an amino-oxy compound on a targeting molecule. Alternatively anamino-oxy maleimide is reacted with the aldehyde and then the maleimideis attached to a cysteine on a targeting molecule (Toyokuni et al.,2003, Bioconj Chem 14:1253).

In another alternative, the AlF-chelator complexes are attached totargeting molecules through click chemistry. The ligands are firstlabeled with Al¹⁸F or Al¹⁹F as discussed above. The AlF-chelator is thenconjugated to a targeting molecule through a click chemistry reaction.For example, an alkyne NOTA is labeled according to Marik and Stucliffe(2006, Tetrahedron Lett 47:6681) and conjugated to an azide containingtargeting agent.

Radiolabeling of Kits With ¹⁸F in Saline

The ¹⁸F (0.01 mCi or higher) is received in 200 μL of saline in a 0.5 mLsyringe and the solution is mixed with 200 μL of ethanol and injectedinto a lyophilized kit as described above. The solution is heated in thecrimp sealed container at 100-110° C. for 15 min. The solution isdiluted with 3 mL water and eluted through an HLB cartridge. Thereaction vial and the cartridge are washed with 2×1 mL portions of waterand then the product is eluted into a vial containing buffered ascorbicacid using 1:1 ethanol water in 0.5 mL fractions. Some of the ethanolmay be evaporated off under a stream of inert gas. The solution is thendiluted in saline and passed through a 0.2 m sterile filter prior toinjection.

Example 34 Maleimide Conjugates of NOTA for ¹⁸F Labeling

As discussed above, in certain embodiments a maleimide derivative ofNOTA may be of use for low-temperature labeling of molecules. Anexemplary method of preparing maleimide-derivatized NOTA is discussedbelow. Details are shown in FIG. 15.

Synthesis of Bis-t-butyl-NODA-MPAA NHS ester: (tBu)₂NODA-MPAA NHS ester

To a solution of (tBu)₂NODA-MPAA (175.7 mg, 0.347 mmol) in CH₂Cl₂ (5 mL)was added 347 μL (0.347 mmol) DCC (1M in CH₂Cl₂), 42.5 mg (0.392 mmol)N-hydroxysuccinimide (NHS), and 20 μL N,N-diisopropylethylamine (DIVA).After 3 h DCU was filtered off and solvent evaporated. The crude mixturewas purified by flash chromatography on (230-400 mesh) silica gel(CH₂Cl₂:MeOH:: 100:0 to 80:20) to yield (128.3 mg, 61.3%) of the NHSester. The HRMS (ESI) calculated for C₃₁H₄₆N₄O₈ (M+H)⁺ was 603.3388,observed was 603.3395.

Synthesis of NODA-MPAEM: (MPAEM=methyl phenyl acetamido ethyl maleimide)

To a solution of (tBu)₂NODA-MPAA NHS ester (128.3 mg, 0.213 mmol) inCH₂Cl₂ (5 mL) was added a solution of 52.6 mg (0.207 mmol)N-(2-aminoethyl) maleimide trifluoroacetate salt in 250 μL DMF and 20 μLDIEA. After 3 h the solvent was evaporated and the concentrate wastreated with 2 mL TFA. The crude product was diluted with water andpurified by preparative RP-HPLC to yield (49.4 mg, 45%) of the desiredproduct. HRMS (ESI) calculated for C₂₅H₃₃N₅O₇ (M+H)⁺was 516.2453,observed was 516.2452.

¹⁸F-Labeling of NODA-MPAEM

To 10 μL (20 nmol) 2 mM NODA-MPAEM solution was added 5 μL 2 mM AlCl₃,40 μL ¹⁸F solution [2.41 mCi, Na¹⁸F, PETNET] followed by 50 μL CH₃CN andheated to 110° C. for 15 minutes. The crude reaction mixture waspurified by transferring the resultant solution into a OASIS® HLB 1 cc(30 mg) cartridge and eluting with DI H₂O to remove unbound ¹⁸F followedby 1:1 EtOH/H₂O to elute the ¹⁸F-labeled peptide. The crude reactionsolution was pulled through the HLB cartridge into a 5 mL vial and thecartridge washed with 3×1 mL fractions of DI H₂O (49.9 The HLB cartridgewas then placed on a new 3 mL vial and eluted with 4×200 L 1:1 EtOH/H₂Oto collect the labeled peptide (0.978 mCi). The reaction vessel retained7.73 pEi, while the cartridge retained 22.1 μCi of activity. 0.978 mCi

=92.5% of Al¹⁸F(NODA-MPAEM).

Example 34 Preparation and Labeling of hMN14 F(ab′)₂

Pepsin Digestion

To 90 ml of 9.8 mg/ml hMN14 IgG C/N 0206078 (Total 882 mg), added 9.0 mlof 0.1M Citrate, pH 3.5 buffer, adjusted to pH 3.7 with 1M citric acid,added 1.2 ml of 1 mg/ml pepsin in 0.1M citrate, pH 3.5 buffer (finalconc. of pepsin was 12 μg/ml). The mixture was incubated at 37° C. for40 min. Digestion was monitored by SE-HPLC and the reaction was stoppedby adding 10% of 3M Tris, pH 8.6

Purification

Was performed on a protein A column (2.5×8 cm, 40 mL). The flowthrough(150 ml) was collected, containing an unbound F(ab′)₂ peak. The F(ab′)₂was filetered through a 0.22 um filter. The final concentration was 15mg/ml (33 ml, total 500 mg). A second step involved Q-SEPHAROSE® columnchromatography (1×20, 16 mL). The protein A purified hMN14 F(ab′)₂ wasconcentrated and dialfiltered into 0.02 M Tris, 0.01M NaCl, pH 8.5, witha 30K stir cell. The F(ab′)₂ was loaded onto a Q-SEPHAROSE® column. Theflow-through peak was collected and fractions 3 and 4 were pooled,concentrated and dilfiltered into 0.04 M PBS, pH 7.4. After filtrationusing a 0.22 um filter, the final concentration was 5.5 mg/ml, 40 mL,total 220 mg of protein.

Preparation of Lyophilized hMN14 Fab′SH

TCEP reduction was performed as follows. To 3.8 mL of 5.5 mg/mLhMN14F(ab′)₂ (total 20.9 mg) was added 0.38 mL of 20 mM TCEP-HCl dilutedin PBS. The mixture was incubated at room temp for 2 hours and thereduction was monitored by SE-HPLC. The Fab′SH was concentrated anddialfiltered into formulation buffer (0.025M NaOAc, 5% trehalose, pH6.7) with a 10K stir cell. About 80 mL buffer was used. The antibody wasfiltered through a 0.22 um filter. The final concentration was 2.3 mg/mL(9 mL, total 20.7 mg). The thiol content per antibody was determined byElman's assay to be 2.4 SH/Fab. Lyophilization was performed using 0.45mL (1 mg protein) per vial.

¹⁸F-Labeling

The lypophilized hMN-14 Fab′ was dissolved in 100 μL 20 mM PBS, pH 7.01.NODA-MPAEM was labeled with 20 nmol of ¹⁸F. Then 600 μL of F-18NODA-MPAEM in 1:1 EtOH/H₂O was added to the Fab′ vial and the mixturewas incubated at RT for 10 min., then purified on a SEPHADEX® G50-80spin column. About 80% of the added activity (80% yield) was eluted froma spin column as labeled antibody. Almost all the activity shifted bySEC after addition of CEA, indicating that the label was associated withthe antibody Fab′ fragment.

What is claimed is:
 1. A method of labeling a molecule with ¹⁸F or ¹⁹Fcomprising attaching a complex of ¹⁸F or ¹⁹F and a group IIIA metal to achelating moiety, wherein the chelating moiety is conjugated to themolecule or the chelating moiety is later attached to the molecule.
 2. Amethod of labeling a molecule with ¹⁸F or ¹⁹F comprising a) attaching acomplex of ¹⁸F or ¹⁹F and a group IIIA metal to a chelating moiety; andb) attaching the chelating moiety to the molecule.
 3. The method ofclaim 1, wherein the molecule is a protein or peptide.
 4. The method ofclaim 1, wherein the molecule is a targeting molecule selected from thegroup consisting of an antibody, a monoclonal antibody, a bispecificantibody, a multispecific antibody, an antibody fusion protein and anantigen-binding antibody fragment.
 5. The method of claim 1, wherein thechelating moiety is conjugated to a targetable construct.
 6. The methodof claim 5, further comprising: a administering a targeting molecule toa subject; b allowing sufficient time for the targeting molecule to bindto a target antigen; and c subsequently administering the targetableconstruct to the subject, wherein the targetable construct binds to thetargeting molecule.
 7. The method of claim 1, wherein the group IIIAmetal is selected from the group consisting of aluminum, gallium,indium, lutetium, and thallium.
 8. The method of claim 1, wherein thegroup IIIA metal is aluminum.
 9. The method of claim 1, wherein thechelating moiety is selected from the group consisting of DOTA, TETA,NOTA, NODA, (tert-Butyl)₂NODA, NODA-MPAA, NETA, C-NETA, L-NETA, andS-NETA.
 10. The method of claim 5, wherein the targetable construct isselected from the group consisting of IMP 448, IMP 449, IMP 460, IMP461, IMP 462, IMP 465, IMP 466, IMP 467, IMP 468, IMP 469, IMP 470, IMP471, IMP 473, IMP 479, IMP 485, IMP 486 and IMP
 487. 11. The method ofclaim 1, wherein the ¹⁸F or ¹⁹F labeled molecule is produced in lessthan 30 minutes from the start of the method.
 12. The method of claim 6,further comprising using PET scanning to image the distribution of the¹⁸F-labeled molecule in the subject.
 13. The method of claim 1, whereinthe ¹⁸F or ¹⁹F complex is attached to the chelating moiety in an aqueousmedium.
 14. The method of claim 1, wherein an organic solvent is addedto the medium to attach the ¹⁸F or ¹⁹F complex to a chelating moiety.15. The method of claim 1, wherein the ¹⁸F or ¹⁹F complex is attached tothe chelating moiety by microwave irradiation.
 16. The method of claim1, wherein the ¹⁸F or ¹⁹F complex is attached to the chelating moiety byheating.
 17. The method of claim 6 wherein the targeting molecule isprepared by the dock-and-lock technique.
 18. The method of claim 17,wherein the targeting molecule is TF2 or TF10.
 19. The method of claim2, wherein the chelating moiety is attached to the molecule by a clickchemistry reaction.
 20. The method of claim 2, wherein the chelatingmoiety is attached to the molecule by a maleimide-sulfhydryl reaction.21. The method of claim 5, wherein the method provides a yield ofradiolabeled targetable construct of is at least 85%.
 22. The method ofclaim 5, wherein the specific radioactivity of the targetable constructis at least 115 GBq/μmol.
 23. The method of claim 5, wherein thetargetable construct is lyophilized for storage before radiolabeling.24. The method of claim 23, wherein the targetable construct islyophilized in a bulking agent selected from the group consisting ofsorbitol, mannitol and trehalose.
 25. The method of claim 1, wherein thechelating moiety is attached to the ¹⁸F or ¹⁹F complex at a pH between3.8 and 4.5.
 26. The method of claim 6, wherein the targeting moleculebinds to a diseased cell or a pathogen.
 27. The method of claim 26,wherein the disease is selected from the group consisting of cancer, acardiovascular disease, an infectious disease, an inflammatory disease,an autoimmune disease, an immune dysfunction disease, graft versus hostdisease, organ transplant rejection and a neurological disease.
 28. Themethod of claim 27, wherein the targeting molecule binds to an antigenselected from the group consisting of carbonic anhydrase IX, CCCL19,CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, CXCR4, CXCR7, CXCL12, HIF-1-α, AFP, PSMA,CEACAM5, CEACAM-6, c-met, B7, ED-B of fibronectin, Factor H, FHL-1,Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible factor (HIF),insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R,IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17,IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUC5, NCA-95, NCA-90, Ia, EGP-1, EGP-2, HLA-DR, tenascin,Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens,tumor necrosis antigens, TNF-.alpha., TRAIL receptor (R1 and R2), VEGFR,EGFR, P1GF, complement factors C3, C3a, C3b, C5a, and C5.
 29. The methodof claim 26, wherein the pathogen is selected from the group consistingof fungi, viruses, parasites, bacteria, human immunodeficiency virus(HW), herpes virus, cytomegalovirus, rabies virus, influenza virus,hepatitis B virus, Sendai virus, feline leukemia virus, Reovirus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus, wart virus, blue tongue virus, Streptococcusagalactiae, Legionella pneumophila, Streptococcus pyogenes, Escherichiacoli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus,Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes,Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus,Mycobacterium tuberculosis and Clostridium tetani.
 30. The method ofclaim 27, wherein the autoimmune disease is selected from the groupconsisting of immune-mediated thrombocytopenia, acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sjogren's syndrome, multiple sclerosis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis, Addison's disease,rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans, primarybiliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,chronic active hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis.
 31. The method of claim 26,wherein the targeting molecule is an antibody selected from the groupconsisting of hR1 (anti-IGF-1R), hPAM4 (anti-pancreatic cancer mucin),hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1(anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR),hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1) andhMN-3 (anti-CEACAM6).
 32. A method of labeling a molecule with ¹⁸F or¹⁹F comprising: a) attaching a group IIIIA metal to a chelating moietyin aqueous medium to form a metal-chelating moiety complex, wherein thechelating moiety is conjugated to the molecule; and b) adding ¹⁸F or ¹⁹Fto the metal-chelating moiety complex, wherein the ¹⁸F or ¹⁹F binds tothe metal.
 33. The method of claim 32, wherein the chelating moiety isNODA-MPAA.